© 2001 Oxford University PressHuman Molecular Genetics, 2001, Vol. 10, No. 21 2385–2396
Comparison of Pkd1-targeted mutants reveals that
loss of polycystin-1 causes cystogenesis and bone
Weining Lu, Xiaohua Shen, Anna Pavlova, Montaha Lakkis, Christopher J. Ward3,
Lynn Pritchard4, Peter C. Harris3, David R. Genest1, Antonio R. Perez-Atayde2 and Jing Zhou*
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
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 aggres-
sive, 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
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
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 extra-
cellular 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 poly-
cystin-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 poly-
cystin-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 identi-
fied (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 develop-
mentally 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 muta-
tion 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
*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: email@example.com
Weining Lu, Genetics Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
2386 Human Molecular Genetics, 2001, Vol. 10, No. 21
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.
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 homo-
logous recombination in embryonic stem (ES) cells (Fig. 1A).
This insertion should result in a frame shift, generating a short-
ened peptide of 130 amino acids which lacks 97% of the poly-
cystin-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.
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 tran-
script 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 deter-
mine 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 poly-
cystin-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 prox-
imal tubules dilated prior to collecting tubules.
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 back-
ground appear to be milder than that in C57/BL-129 back-
ground 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 aggres-
sive 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).
Human Molecular Genetics, 2001, Vol. 10, No. 21 2387
Figure 1. Targeted disruption of exon 4 of Pkd1 and differential expression of Pkd1 in null and del34 mutants. (A) Structures of wild-type and targeted Pkd1 null
alleles. A phosphoglycerate kinase (pgk) promoter-driven neo-resistant gene was inserted into exon 4 of Pkd1. Single arrows indicate the direction of transcription.
A, AvrII; B, BamHI; N, NsiI; P, PmlI. F27, R25 and R37 are primers used to detect transcripts shown in (C). (B) Southern analysis of targeted mouse mutants. DNA
isolated from F2 null littermates was digested with BamHI and hybridized with a 1 kb genomic fragment [probe 2 in (A), see Materials and Methods for probe 1
information]. The 3.7 and 5.4 kb bands correspond to wild-type and mutant alleles, respectively. (C) RT–PCR of Pkd1 transcripts in the wild-type (+/+), hetero-
zygous (null/+) and homozygous (null/null) whole mouse embryos using primers F27, R25 and R37, whose positions are shown in (A). C, wild-type control with-
out reverse transcriptase. The band shown in C is an amplification product of wild-type genomic DNA (containing introns 2, 3 and 4) which is larger than the cDNA
shown in the (+/+) lane. (D) Northern analysis of mRNA from E12.5 whole embryos was probed with a Pkd1 cDNA probe (exons 2–6). An ∼14 kb Pkd1 transcript
in wild-type (+/+), heterozygous (null/+), and homozygous (null/null) embryos was seen. A mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe
was used as a loading control. The intensity of the 14 kb band in all genotypes after normalization with GAPDH is shown in a chart on the right. (E) Northern
analysis of mRNA from E12.5 whole mouse embryos. The same Pkd1 cDNA probe used in (D) detects a full-length 14 kb Pkd1 transcript in del34 mutants and in
wild-type. An additional 11 kb transcript was detected in del34 heterozygotes and in del34 homozygotes. Mouse GAPDH was used as a loading control. The intensity of
the 11 and 14 kb bands in all genotypes after normalization with GAPDH is shown in a chart on the right. (F) Western analysis of membrane fractions from E12.5 whole
mouse embryos. A monoclonal antibody (7e12) against the N-terminus of polycystin-1 detects a prominent ∼460 kDa band in wild-type (+/+) and heterozygotes (null/+)
and is absent in the homozygous (null/null) mutants. (G) Western analysis of E16.5 whole mouse embryos with the same polycystin-1 antibody used in (F), 7e12.
An ∼460 kDa protein (polycystin-1) was detected in wild-type and del34 heterozygotes. A smaller band, and a nearly full-length band is overexpressed in heterozygotes
and homozygotes compared with wild-type. (H) DNA sequence of the RT–PCR products from null homozygotes (C). The mutant transcript would result in a premature
stop codon (tag) immediately beyond exon 3 whose sequence is underlined.
2388 Human Molecular Genetics, 2001, Vol. 10, No. 21
Kidney, liver and pancreatic cystic disease in
Pkd1-targeted heterozygous mice
Among 40 null heterozygotes (2–24 months) examined, micro-
scopic 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 hetero-
zygotes developed visible renal cysts (at 13, 19.5, 21, 22.5 and
23 months old); three out of five had bilateral cysts.
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 homo-
zygotes, 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 struc-
tures 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).
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 back-
ground, 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.
Spina bifida occulta and osteochondrodysplasia in Pkd1
Cartilage and bone develop from multiple embryological
origins. The craniofacial skeleton in the first branchial arch and
Table 1. Number of embryos examined from Pkd1null/+ intercrosses at various developmental stages
Numbers in parenthesis indicate null homozygotes that have been examined histologically for renal cystic lesions. All embryos >E15.5 were grossly examined for
the size of their heart, kidney and lungs, and the size and visibility of cysts in their kidney, pancreas and liver.
Embryonic day Total embryos
Table 2. Kidney and liver cystic lesions in Pkd1null/+ mice
aMicroscopic renal cysts found in seven out of 10 mice at 2–24 months of age.
bMacroscopic liver cysts found in 12 out of 25 mice at 2.5–16 months of age.
Age (months) 2.5
Age (months) 11
Human Molecular Genetics, 2001, Vol. 10, No. 21 2389
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.
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
Figure 2. Renal and pancreatic phenotype in null and del34 homozygous kidney. (A and B) Initial stage of cystic lesions in kidneys at E15.5 in C57BL/6-129
background null/null and del34/del34 mutants (H&E; magnification 50×). Note the severe disease in null homozygotes. (C and D) Cystic kidneys at E17.5 in
C57BL/6-129 background in null/null and del34/del34 mutants (H&E; magnification 50×). (E and F) Cystic kidneys at newborn stage in BALB/c-129 background
null/null and del34/del34 mutants (H&E; magnification 50×). Note that the degree of cystic lesion is similar to that in C57BL/6-129 background at E17.5. (G and
H) Pancreatic cysts at E15.5 in C57BL/6-129 background null/null and del34/del34 mutants (H&E; magnification 100×). (I and J) Pancreatic cysts at E18.5 in
BALB/c-129 background null/null and del34/del34 mutants (H&E; magnification 50×).
2390 Human Molecular Genetics, 2001, Vol. 10, No. 21
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, reminis-
cent of human infantile osteodystrophy. These data suggest
that polycystin-1 is required for the differentiation of chondro-
cytes. Its loss leads to defects in the development of the verte-
bral column, skeletal growth and ossification including
Polycystin-1 is required in the final stages of kidney
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 distri-
bution 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 morpho-
genesis 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
Figure 3. Pancreatic cyst phenotype in a del34 heterozygote. (A) Pancreatic cysts (c) from a 22-month-old del34 heterozygous mouse. du, duodenum; pa,
pancreatic tissue; sp, spleen; st, stomach. (B) Multiple pancreatic cysts (c) lined by a monolayer of cuboidal epithelia and thin fibrous wall (H&E; magnification
50×). (C) Higher magnification of cyst-lining cuboidal epithelia. Remaining acini can be seen in the lower left corner (H&E; magnification 200×). (D) Pancreatic
cysts (c) with eosinophilic cuboidal epithelium (H&E; magnification 200×). (E) Atrophy of exocrine pancreas with cyst formation, focal fibrosis and adipose
replacement. Few acini (arrow) and islets (arrowhead) remain (H&E; magnification 50×). (F) In more solid areas, the pancreas is massively replaced by adipose tissue
(pancreatic lipomatosis); atrophic acini (arrow) and islets of Langerhans (arrowhead) appear isolated and surrounded by mature adipocytes (H&E; magnification 50×).
Table 3. Newborn Pkd1null/null and Pkd1del34/del34 mice with skeletal defects
++, Severe; +, mild; –, negative.
Human Molecular Genetics, 2001, Vol. 10, No. 21 2391
become fully differentiated tubules in permanent kidneys. This
process can be classified into four stages: nephrogenic conden-
sation, 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 mesen-
chyme 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, coin-
cident 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 struc-
tural integrity of renal tubules. Although glomerular expres-
sion 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
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 poly-
cystin-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 overex-
press 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
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 back-
ground. 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 alterna-
tive spliced transcripts, alternative splicing in other parts of the
Pkd1 gene can not be excluded. On the other hand, the alterna-
tive 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 initi-
ates clonal cyst development in ADPKD.
Polycystin-1 is required for skeletogenesis
Both homozygous Pkd1-targeted mutants exhibit osteochon-
drodysplasia and delayed endochondral and intramembranous
bone formation, demonstrating that polycystin-1 is required for
Figure 4. Polyhydroamnios and hydrops in del34 and null homozygotes. (A) E14.5
null homozygote (null/null) and wild-type (+/+) embryos with intact
amniotic sac and placenta (p). Excess amniotic fluid was found inside the
amniotic sac of the null homozygote. (B) The amount of amniotic fluid from
E14.5 embryos of the same litter with different genotypes. (C) Massive
systemic edema in an E15.5 null homozygote compared with a wild-type
littermate. (D) Hydrops at a lesser degree was found in del34 newborn homo-
zygotes (del34/del34). Normal heterozygote (del34/+) and wild-type (+/+)
littermates are shown on either side of the homozygote. (E) Massive subcuta-
neous edema (arrow) in a cross-section of an E15.4 null homozygote (H&E;
magnification 25×). (F) Cross-section of a wild-type littermate of (E) shows
normal subcutaneous tissue (arrow) (H&E; magnification 25×).
2392 Human Molecular Genetics, 2001, Vol. 10, No. 21
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 poly-
cystin-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 hetero-
zygous 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
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 excel-
lent models for studies of both the pathogenesis of ADPKD
and polycystin functions.
MATERIALS AND METHODS
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
Figure 5. Pkd1 transcript expression in developing vertebrae and long bone. (A) Scheme of lumbar vertebrae in a normal newborn mouse. The enlarged views of
the boxed area are in (D and E). (B) Overview of strong expression of Pkd1 in the lumbar vertebrae (magnification 50×) from a newborn mouse. (C) H&E staining
of the section adjacent to (B) (magnification 50×). (D) High expression of Pkd1 in the perichondrium of vertebral arch joint (magnification 100×). (E) Phase
contrast of (D) (magnification 100×). (F) High expression of Pkd1 in the perichondrium of distal part of femur from E16.5 embryo. Lower levels of expression can
be seen in the resting chondrocytes through prehypertrophic chondrocytes and absent in the hypertrophic chondrocytes (arrow; magnification 50×).(G) Toluidine
blue staining of the section adjacent to (F) (magnification 50×).
Human Molecular Genetics, 2001, Vol. 10, No. 21 2393
exons 2–6 was subcloned into vector litmus 28. A phos-
phoglycerate 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 electro-
porated 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
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. Hetero-
zygotes 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 frag-
ment 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
Figure 6. Spinal bifida and osteochondrodysplasia in Pkd1 homozygous mutants. Newborn mice were stained with alizarin red for bone (red color) and alcian blue
for cartilage (blue color), followed by alkaline digestion to allow visualization of the skeleton. Genotypes of the mice are labeled. (A) Ventrodorsal view of intact
skeletons showing dwarfism and defective ossification in the homozygous mutants. (B–D) View of lumbar region vertebrae (arrow). Note the wide separation of
the neural arches in null/null, mild separation in del34/del34, and complete closure of neural arches in wild-type (+/+). (E and F) Close view of null homozygous
lumbar vertebrae showing the opening of neural arches (arrow) (E) compared with normal wild-type littermate (F). Ossification is initiated but to a lesser degree
in the null homozygotes. (G and H) Comparison between forelimbs (G) and hindlimbs (H) of a null homozygote and wild-type. (I–K) The craniofacial skeleton
of a newborn null homozygote (null/null) and its wild-type littermate (+/+); note the delayed mineralization in null/null. (L) Dorsal view of the laryngeal
cartilages; note the defect of the thyroid cartilage (arrow) in mutants.
2394 Human Molecular Genetics, 2001, Vol. 10, No. 21
the mutant fragment is 14.8 kb (data not shown). The homo-
logous 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
(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
charged nylon membranes
US National Institutes of Health and available on the internet
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 frag-
ments 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.
Figure 7. Histological analysis of endochondral ossification in Pkd1 null/null mutants. (A and B) Longitudinal sections of wild-type (+/+) and mutant (null/null)
humerus from E14.5 embryos. Note the reduced hypertrophic chondrocyte zone in mutant (arrow bar) (H&E; magnification 50×). (C and D) High magnification
of chondrocytes in the center of bones shown in (A and B). Calcium deposits can be easily found in wild-type (arrow), but not in the mutant. (H&E; magnification
50×). (E and F) Longitudinal sections of wild-type (+/+) and mutant (null/null) radius from E17.5 embryos. The bone marrow cavity is formed but slightly
reduced (arrow bar) (H&E; magnification 50×) in the mutant. (G and H) High magnification of proliferating zone of bones shown in (E) and (F) (H&E;
Human Molecular Genetics, 2001, Vol. 10, No. 21 2395
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 ribo-
probe 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
Antibodies and western blot analysis
Mouse monoclonal antibody 7e12 was raised against an
epitope in the N-terminal flank-LRR-flank domain of human
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 poly-
cystin-1 at 1:1000. Bound protein was detected by enhanced
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 evis-
cerated 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.
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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