Corrections

MEDICAL SCIENCES. For the article ‘‘Essential role of cleavage ofPolycystin-1 at G protein-coupled receptor proteolytic site forkidney tubular structure,’’ by Shengqiang Yu, Karl Hackmann,Jianggang Gao, Xiaobing He, Klaus Piontek, Miguel A. GarcıaGonzalez, Luis F. Menezes, Hangxue Xu, Gregory G. Germino,Jian Zuo, and Feng Qian, which appeared in issue 47, November20, 2007, of Proc Natl Acad Sci USA (104:18688–18693; firstpublished November 14, 2007; 10.1073�pnas.0708217104), theauthors note that the author name Jianggang Gao should haveappeared as Jiangang Gao, and the author name Miguel A.Garcıa Gonzalez should have appeared as Miguel A. Garcıa-Gonzalez. The online version has been corrected. The correctedauthor line appears below.

Shengqiang Yu, Karl Hackmann, Jiangang Gao, XiaobingHe, Klaus Piontek, Miguel A. Garcıa-Gonzalez, Luis F.Menezes, Hangxue Xu, Gregory G. Germino, Jian Zuo,and Feng Qian

www.pnas.org�cgi�doi�10.1073�pnas.0711495105

MICROBIOLOGY. For the article ‘‘The P2 capsid protein of thenonenveloped rice dwarf phytoreovirus induces membrane fu-sion in insect host cells,’’ by Feng Zhou, Yingying Pu, TaiyuanWei, Huijun Liu, Wulan Deng, Chunhong Wei, Biao Ding,Toshihiro Omura, and Yi Li, which appeared in issue 49,December 4, 2007, of Proc Natl Acad Sci USA (104:19547–19552;first published November 27, 2007; 10.1073�pnas.0708946104),the authors note that the author name Taiyuan Wei should haveappeared as Taiyun Wei. The online version has been corrected.The corrected author line appears below.

Feng Zhou, Yingying Pu, Taiyun Wei, Huijun Liu, WulanDeng, Chunhong Wei, Biao Ding, Toshihiro Omura,and Yi Li

www.pnas.org�cgi�doi�10.1073�pnas.0711787105

1386 � PNAS � January 29, 2008 � vol. 105 � no. 4 www.pnas.org

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Essential role of cleavage of Polycystin-1at G protein-coupled receptor proteolyticsite for kidney tubular structureShengqiang Yu*†, Karl Hackmann*, Jiangang Gao‡, Xiaobing He*, Klaus Piontek*, Miguel A. Garcıa-Gonzalez*,Luis F. Menezes*, Hangxue Xu*, Gregory G. Germino*, Jian Zuo‡, and Feng Qian*§

*Department of Medicine, Division of Nephrology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and ‡Department of DevelopmentalNeurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105

Edited by Mark T. Keating, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, and approved October 5, 2007 (received for reviewAugust 30, 2007)

Polycystin-1 (PC1) has an essential function in renal tubular mor-phogenesis and disruption of its function causes cystogenesis inhuman autosomal dominant polycystic kidney disease. We havepreviously shown that recombinant human PC1 is cis-autoproteo-lytically cleaved at the G protein-coupled receptor proteolytic sitedomain. To investigate the role of cleavage in vivo, we generatedby gene targeting a Pkd1 knockin mouse (Pkd1V/V) that expressesnoncleavable PC1. The Pkd1V/V mice show a hypomorphic pheno-type, characterized by a delayed onset and distal nephron segmentinvolvement of cystogenesis at postnatal maturation stage. Weshow that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist.Our study establishes a critical but restricted role of cleavage forPC1 function and suggests a differential function of the two typesof PC1 molecules in vivo.

autosomal dominant polycystic kidney disease �cis-autoproteolytic cleavage � GPS � knockin mouse � tubulogenesis

Polycystin-1 (PC1) is the product of PKD1, the principal genemutated in human autosomal dominant polycystic kidney

disease (ADPKD) affecting 1 of 1,000 people (1). The disease ischaracterized by the progressive development of multiple bilat-eral cysts in the kidney, resulting in renal failure in 50% ofpatients by their sixth decade of age (2). ADPKD is a systemicdisease with many extrarenal manifestations including hepaticand pancreatic cysts and intracranial aneurysms.

Pkd1 knockouts exhibit a variable degree of early extrarenaldevelopmental abnormalities including polyhydramnios, s.c.edema, and hemorrhage, but invariably develop severe polycystickidneys, culminating in embryonic lethality (3–6). This findingindicates wider-ranging functions of PC1 in embryonic devel-opment. PC1 (mouse) is a 4,293-aa 11-transmembrane (TM)glycoprotein with a 3,066-aa-long N-terminal ectodomain thatcontains a unique combination of motifs involved in protein–protein interactions, and a �200-aa cytoplasmic C terminus (7,8) that can activate a number of signaling pathways (9). Theectodomain is separated from the 11-TM segment by the Gprotein-coupled receptor (GPCR) proteolytic site (GPS) do-main of �50 aa (10). The GPS domain was first identified as aninternal cleavage site in a neuronal GPCR protein CL and waslater found at a similar position in �30 adhesion GPCRs that areextraordinary for having an unusually large and complex ectodo-main (reviewed in ref. 11). We have shown that recombinanthuman PC1 is cleaved at HLˆT (where “ˆ” identifies the positionof cleavage) within the GPS domain in early secretory pathwaythrough cis-autoproteolysis (12, 13). The cleavage results in theN-terminal fragment (NTF) and the 11-TM C-terminal fragment(CTF) that remain noncovalently tethered to each other (12).Cleavage is incomplete, with similar ratios of the uncleavedfull-length uFL-PC1 and the cleaved PC1 products. PKD1-associated missense mutations in the vicinity of the cleavage site

disrupt cleavage and abolish PC1’s ability to activate the Jak2-Stat pathway and to induce in vitro tubulogenesis in MDCK cells(12). The uFL-PC1 and cleaved PC1 products therefore mayhave different functions in vivo.

Proteolytic cleavage is a common regulatory mechanism forprotein functions (14). It has recently been reported that PC1undergoes cleavage by a non-GPS-mediated process to release afragment from its cytoplasmic C terminus from the membranein association with mechanical stimuli, and this fragment entersthe nucleus to initiate signaling (15, 16). However, the mecha-nism by which GPS cleavage affects PC1 function is unknown.The GPS sequence is present in the entire PC1 family (13), butsome members such as PKDREJ (17) and suREJ2 (18) are, infact, not cleaved in vivo. In addition, most previous studies havereported that endogenous PC1 is not cleaved at GPS in variouscell types and tissues including the kidney (e.g., refs. 19–21).These findings raise a question about the functional significanceof GPS cleavage for PC1 in vivo.

In the present study, we generated and characterized a Pkd1knockin mouse without GPS cleavage of PC1 to examine thefunction of the cleavage in vivo. We analyzed the cleavagepatterns of endogenous PC1 in WT and mutant mice. Our studyestablishes a critical but restricted role of cleavage for PC1function in vivo.

ResultsGeneration of Pkd1 Knockin Mouse with Absent GPS Cleavage of PC1.We chose a reverse genetic approach, mutating the codon ACT(Thr) to a GTT (Val) at amino acid position 3041 of the cleavagesite HLˆT3041 in the mouse Pkd1 gene to investigate the functionof PC1 cleavage in vivo. The mutation T3041V is known toprevent cleavage of recombinant PC1 (13) and is expected todisrupt presumed cleavage of PC1 in the mouse. The resultingnoncleavable mutant PC1 (named PC1V) should direct a Pkd1null-like phenotype if cleavage is essential for PC1 function.

We generated and transfected the targeting vector (Fig. 1A i)into ES cells for homologous recombination and identified 1 of200 selected ES clones that had proper targeting (Fig. 1B). Thisclone (G8) was used to generate F1 heterozygous Pkd1NeoV/�

mice (Fig. 1 A iii), which appeared normal and were fertile. Theirintercross did not produce viable homozygous offspring, prob-

Author contributions: F.Q. designed research; S.Y., K.H., J.G., X.H., H.X., and J.Z. performedresearch; G.G.G. and F.Q. contributed new reagents/analytic tools; S.Y., K.H., K.P.,M.A.G.-G., L.F.M., and F.Q. analyzed data; and F.Q. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

†Present address: Division of Nephrology, Shanghai Changzheng Hospital, Second MilitaryMedical University, Shanghai, 200003, People’s Republic of China.

§To whom correspondence should be addressed. E-mail: fqjhupkd@jhmi.edu.

© 2007 by The National Academy of Sciences of the USA

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ably because of interference of RNA splicing by Neo (data notshown). We therefore removed Neo by crossing the Pkd1NeoV/�

mice with FLP transgenic mice (22) (Fig. 1C), as confirmed bygenomic DNA sequencing (data not shown). The resultingPkd1V/� heterozygotes (Fig. 1 A iv) lived as long as the WTlittermates did and showed no cysts in the kidney and liver, orany other abnormalities, at 13 and 22 months (n � 6).

Pkd1V/� mice exhibited normal reproduction when inter-crossed, with typical litter size between 6 and 10 pups. Pkd1V/V

homozygotes were born at the predicted Mendelian frequencywhen examined in a total of 255 offspring (Table 1). This resultshows that the Pkd1V allele can overcome embryonic lethalityusually seen in the Pkd1�/� mice. This finding was confirmed bythe normal appearance of Pkd1V/V embryos (E15; n � 3) as theWT littermates (data not shown). We confirmed the propermRNA expression of the Pkd1V allele in the mouse by RT-PCR.Only a single product of predicted size for WT mRNA wasamplified from the kidneys of postnatal day 9 (P9). Pkd1V/V,Pkd1V/�, and WT littermates with primers within exon 23 and

exon 27 (Fig. 1D) and each product showed the expectedsequence at codon 3041 (Fig. 1E). The same result was obtainedfor liver, spleen, brain, lung, and heart (data not shown).

Cystic Kidney Development in Postnatal Pkd1V/V Mice. The Pkd1V/V

mice appeared normal in the first 8 days of postnatal life, butbecame slightly smaller than the WT or Pkd1V/� littermates fromP9 onward (Fig. 2A). By P16, all animals displayed markedlysmaller body statures with distended abdomens (Fig. 2B). Micekilled at P9 and P16 had pale, grossly enlarged cystic kidneys(Fig. 2 A and B Right), as well as dilated common bile ducts (datanot shown). At 3 weeks or after, the Pkd1V/V mice weighed lessthan half the WT or Pkd1V/� littermates, but had a considerablylarger kidney/body ratio (26.9 � 2.1, n � 3) than the WT orPkd1V/� littermates (1.3 � 0.1, n � 8). They died between 2 and6 weeks after birth, with �50% dead by the third week of age(Fig. 2C), presumably because of renal insufficiency as indicatedby the elevated blood urea nitrogen (BUN) levels (Fig. 2D).Pancreas and spleen appeared normal.

Tubular Cystic Dilation in Pkd1V/V Mouse Kidney. Histological exam-ination revealed a rapid and progressive tubular dilation of thePkd1V/V kidneys during the postnatal maturation stage (Fig. 3).At P0, the mutant kidneys contained a few microcysts (�10discrete cysts) in the subcortical region, but otherwise appearednormal (Fig. 3A). From P1 through P5, both the number and sizeof the cysts gradually increased, initially in the cortex and thenextending into the medulla. The overall kidney sizes, however,were not noticeably enlarged as compared with those of the WTlittermates, and significant amounts of normal parenchymaremained (Fig. 3 A and B).

Cystic dilation extended progressively into the medulla fromP7 onward, and by P14, cysts had replaced most of the normalrenal parenchyma with the exception of the papillae tip thatremained grossly intact (Fig. 3A). Intact glomeruli and tubuleswith brush borders characteristic of proximal tubules (PTs) werereadily identified in the cortical region at this stage (Fig. 3 C andD). Both structures were consistently observed throughout laterstages, at P22 (Fig. 3E) and P28 (Fig. 3 E and F Inset), whenmassively cystic kidneys reached a length of �15 mm and aweight of �10 times the kidney of the WT littermate and intactpapillae were no longer observed.

The Pkd1V/V mice also showed mild dilation of biliary ducts

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Fig. 1. Generation of the Pkd1V/V mouse by gene targeting. (A) Structure ofvarious Pkd1 alleles. (i) Targeting vector. Neo, FRT-flanked (red triangle)PGK-neomycin selection cassette; TK, thymidine kinase gene; S, SpeI. Exons aredepicted by solid boxes. The T3041V mutation is indicated. (ii) Pkd1 WT allele,with exon number shown. (iii) Pkd1NeoV allele. The expected size of the SspI orSpeI restriction fragments for WT and Pkd1NeoV alleles by using the 5� probe(within exon 15 outside of the vector) is shown. The expected size of the 3�long-range PCR product is also shown. (iv) Pkd1V allele. A single FRT site of 47bp is retained at the site of Neo insertion. (B) Genomic Southern blot showingcorrect targeting of the ES clone G8 by detection of 9.9-kb (SspI) and 10.4-kb(SpeI) Pkd1NeoV-specific bands by the 5� probe. The 3� long-range genomic PCRconfirmed correct targeting (data not shown). (C) Removal of Neo in theoffspring of the Pkd1NeoV/�:FLP mating as shown by the three-primer PCR-based strategy. (i) Primer positions and the expected size of the PCR productof the each allele. (ii) PCR result showing successful removal of Neo byamplification of the Pkd1V-specific 329-bp product (Upper) only in the FLP-positive offspring (Lower). �, negative control for PCR. (D) RT-PCR from WT,Pkd1V/� (V/�), and Pkd1V/V (V/V) kidneys at P9. The expected size of the PCRproduct using exon 23 and 27 specific primers is shown (Upper). A singleproduct of expected size was amplified from each genotype (Lower). DNA sizemarker is shown on right. (E) DNA sequence of the RT-PCR products in D at thecleavage size.

Table 1. Genotype ratios in a total of 255 mice obtained from 26interbreedings of heterozygous Pkd1V/� mice

Genotype �/� V/� V/VNumber 65 124 66Actual ratio, % 25.5 48.6 25.8Expected ratio, % 25 50 25

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Fig. 2. Postnatal development of cystic kidney in Pkd1V/V mice. Generalappearance of the mice and their kidneys at P9 (A) and P16 (B). Note thatPkd1V/V kidneys are enlarged, pale, and cystic compared with kidneys from WTand heterozygous (V/�) littermates. (C) Survival curve of WT (n � 33), V/� (n �62), and V/V (n � 29) mice. The median age of survival of Pkd1V/V mice is 23days. (D) Elevated BUN level in the Pkd1V/V mouse as compared with that inPkd1V/� and WT littermates at P14 and P23.

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surrounded with fibrotic areas, starting at P7 through P28(Fig. 3G).

Segments of Origin of Renal Tubular Cysts. We determined cystorigins by using a set of nephron segment-specific markers. AtP1, the few cysts seen were of collecting duct (CD) origin (AQP2positive), whereas PT (LTL positive) was intact (Fig. 4A). At P5,virtually all cysts were of CD origin (DBA positive) in themedulla (Fig. 4 B–D), whereas PT (Fig. 4B) and thick ascendinglimb (TAL) (Fig. 4D, NKCC2 positive) were intact. In the cortex,three types of cysts could be identified: (i) DCT-derived (THPpositive and DBA and NKCC2 negative on serial sections) (Fig.4C and D, *); (ii) CD-derived (DBA positive) (Fig. 4 C and D,arrowhead); (iii) probably derived from the junction betweenDCT and CD (composite staining with DBA and anti-THP)(Fig. 4 C and D, rectangle). At P9, most cysts were eitherCD-derived (Fig. 4 E and F) or, to a lesser extent, DCT-derived(�10%) (Fig. 4E, star), whereas TAL (Fig. 4F Inset) and PT(data not shown) were intact. At P14 (Fig. 4G) and P28 (Fig. 4H),virtually all cysts had a CD origin, whereas PT (Fig. 4 G and H),now confined to the narrow areas between adjacent cystic walls,were consistently intact, thus validating our morphologicalfindings.

Our results show that the renal tubular cystic dilation inPkd1V/V kidney involves primarily distal nephron segments, butnot the more proximal nephron segments, throughout the post-natal stages (Fig. 4I). Our finding contrasts with the findings inPkd1�/� mice (3) or human ADPKD (23), in which all segmentsof the nephron are affected. The Pkd1V allele, therefore, directsa hypomorphic phenotype, with a delayed onset and distalsegment-specific involvement of cystic dilation at postnatalstage.

PC1 Cleavage Pattern During Embryonic Development. To elucidatethe basis of the observed hypomorphic activity of the Pkd1V

allele, we characterized the cleavage pattern of endogenous PC1in the WT and Pkd1V/V mice. We first analyzed PC1 cleavage inmurine embryonic fibroblasts (MEFs) isolated from E12 em-bryos of WT, Pkd1V/V, Pkd1V/�, and Pkd1�/� genotypes, by acombination of immunoprecipitation (IP) and Western blot with

use of anti-CC, a new polyclonal antibody against the C-terminal170 aa of mouse PC1 (Fig. 5 A). The specificity of this antibodyis demonstrated by its detection of both uFL and CTF of theC-terminally FLAG-tagged recombinant PC1 encoded by thefull-length mouse Pkd1 cDNA (mPC1-F) after IP with eitheranti-FLAG or anti-CC (Fig. 5B). This cleavage pattern is iden-tical to that of the equivalent human recombinant PC1 (lanehPKD1-F) by anti-CT, as found (12, 13), and endogenous PC1of the human embryonic kidney cell line (HEK).

We found that PC1 of WT MEF is incompletely cleaved, asevidenced by the detection of both uFL-PC1 of �520 kDa andCTF of �150 kDa (Fig. 5C), a pattern similar to that ofendogenous human PC1 of HEK cells. The identity of thesebands is confirmed by the finding that they were detected in thePkd1V/� MEF, but not in Pkd1�/� MEF. Cleavage of theendogenous PC1 of WT MEF is more extensive than that of therecombinant PC1. In the Pkd1V/V MEF, only the �520-kDaPC1V, and no CTF, was detected. This result indicates thatnoncleavable PC1V was indeed expressed in Pkd1V/V MEF, at alevel 1.5–2 times the uFL-PC1 of WT MEF. This result is furtherconfirmed by positive and negative detection of NTF by anti-LRR in WT and Pkd1V/V MEF, respectively (data not shown).Similar cleavage patterns were found in E12 embryos (Fig. 5D).

PC1 cleavage also occurs in WT embryos at various laterstages (Fig. 1E), when various abnormalities occur in Pkd1�/�

but not in Pkd1V/V mice. This finding shows that PC1V issufficient to overcome embryonic lethality usually seen inPkd1�/� mice, and the GPS-cleaved PC1 products are notessential for embryonic development.

PC1 Cleavage Patterns in Postnatal and Adult Mice. We next exam-ined the cleavage pattern of PC1 in postnatal kidneys, whenrapid tubular cystic dilation is occurring in the Pkd1V/V kidneys.As shown in Fig. 5F, PC1 is extensively cleaved in WT kidneysat P7 and P14, whereas PC1 became undetectable at P21. PC1V

was expressed in the cystic kidneys of P7 and P14 Pkd1V/V

littermates at higher levels than uFL-PC1, but was hardlydetectable at P21. We further demonstrated that cleavage of PC1occurred in the CD cells isolated from P5, P10, and P14 WTkidneys (Fig. 5G). PC1V was expressed in the cystic CD cells

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Fig. 3. Rapid and progressive cystic dilation in Pkd1V/V postnatal kidneys. (A) Overview of Pkd1V/V kidneys of various postnatal stages in H&E-stained sections,with two to three animals analyzed for most of the stages. (Scale bars, 2 mm.) Note the intact papilla tip in P3–P14 kidneys. P22 and P28 kidneys are more enlarged(�15 mm long) with further cystic expansion (data not shown). (B–D) H&E-stained sections of P5, P7, and P14 Pkd1V/V kidneys, respectively. Note the intactglomeruli and proximal tubule (PT)-like structures. (E) Masson-trichrome (MT)-stained kidney section of P22 Pkd1V/V mouse. Note the intact glomeruli and PT-liketubules surrounded by interstitial fibrosis. (F) H&E-stained section of P28 Pkd1V/V kidney. Note the large size of the cysts and intact glomeruli and PT-like tubules(Inset). (G) MT-stained liver section of P14 Pkd1V/V mouse. Note the fibrosis around the dilated biliary ducts.

18690 � www.pnas.org�cgi�doi�10.1073�pnas.0708217104 Yu et al.

isolated from the respective Pkd1V/V littermates at levels com-parable to the WT uFL-PC1 (Fig. 5G), thus excluding a CD-specific down-regulation of the PC1V expression as a trivialexplanation for its cystic changes. This result indicates that PC1V

is not sufficient for the integrity of CD (except the papilla tip)during the postnatal maturation stage and that the cleaved PC1products are most probably required for the process.

PC1 was also cleaved in primary PT cells isolated from P5 andP10 WT kidneys (Fig. 5G), as well as in primary human PT cellsfrom an adult kidney (data not shown). PC1V was expressed inthe PT of the Pkd1V/V littermates at a level similar to theuFL-PC1. This finding indicates that PC1V is sufficient for thenormal structure of PT, whereas the cleaved PC1 is not essential.

We finally analyzed the cleavage patterns in various organs ofWT postnatal and adult mice. We found that PC1 was extensivelycleaved at P3 (Fig. 1H, WT), P7, P14 (data not shown), and P19(Fig. 5I, including spleen and pancreas). Similar cleavage pat-terns were found for the organs of adult mice at postnatal 6weeks and 3 months (data not shown). PC1V was not cleaved atall times at P3 (Fig. 1 H, V/V) or P7 (data not shown), and wasexpressed at a level two to three times the correspondinguFL-PC1. Our results demonstrate that cleavage of endogenousPC1 is a conserved and ubiquitous phenomenon.

DiscussionTo investigate the function of GPS cleavage of PC1 in vivo, wegenerated and characterized the Pkd1V/V mouse without GPS

cleavage. The Pkd1V/V mice show several striking features thatare not seen in Pkd1�/� mice: (i) they are born with normalMendelian frequency and are viable; (ii) they show rapid cysticdilation in CD (except the papilla tip) and DCT, but not in theproximal portion of the nephron, during the postnatal period,and die with severe uremia, mostly at 3 weeks of age; (iii) theyshow dilation of the common bile duct and intrahepatic biliaryducts, but develop a normal pancreas within their life span. Weconclude from these data that cleavage is required for theintegrity of almost all the distal nephron segments, but not forproximal nephron segments, during the postnatal maturationperiod. It is also critical for the normal structure of common bileduct and intrahepatic bile tracts. It is, however, not essential forthe embryonic development of various organ systems includingkidney and pancreas.

To understand the basis of hypomorphic phenotype of thePkd1V/V mice, we characterized PC1 cleavage patterns in WTand mutant mice. We found that endogenous PC1 is ubiquitouslybut incompletely cleaved in WT mice at various developmentalstages, as is endogenous human PC1 in HEK and renal epithelialcells. Cleavage results in two classes of PC1 molecules: a smallamount of uFL-PC1 and the more abundant cleaved PC1products. In the Pkd1V/V mice, the mutant PC1V is not cleavedat all times and is expressed with overall stage- and tissue-specificpatterns similar to uFL-PC1, in general, at two to three times thelevels uFL-PC1. This modestly higher level of PC1V over the WTuFL-PC1 per se is unlikely to be the reason for the cystic changein Pkd1V/V mice, because hemizygous Pkd1V/�mice, with thedosage of PC1V presumably reduced to half, showed a phenotypesimilar to the Pkd1V/V mice (data not shown). PC1V is mostprobably functionally equivalent to uFL-PC1 based on the natureof the T3041V mutation chosen: (i) it is precisely at the �1position of the cleavage site HLˆT of the GPS domain, which isso far known only for the function as a cis-autoproteolytic signal;(ii) Thr and Val differ solely by one functional group at the veryterminus of the side chain (OOH vs. OCH3). Although effec-tively disrupting cleavage of PC1 by preventing the nucleophilicattack, the critical initial step of cis-autoproteolysis (13), thissmallest possible change seems less likely to significantly alter theconformation surrounding the cleavage site or the overall con-formation in PC1V. Unchanged conformation by similar �1mutations is in fact shown for the cis-autoproteolytic protein bystructural analysis (24). Therefore, our knockin approach spe-cifically devoids all GPS-cleaved products, while it preserves theuncleaved form that should represent the WT uFL-PC1. Thisoutstanding feature could make this mouse model very infor-mative in dissecting differential functions of uFL and cleavedPC1 molecules in vivo.

We can conclude from our data that PC1V is sufficient torescue the embryonic lethality and to prevent various abnor-malities during embryonic development such as severe cysticexpansion of kidney and pancreas, usually observed in Pkd1knockouts. Our results suggest that uFL-PC1 plays a critical roleduring embryonic development, whereas the GPS-cleaved PC1products are not essential in this process.

Our data also suggest a differential role of uFL-PC1 andcleaved PC1 molecules for the proximal and distal nephronsegments during postnatal phase. We find that PC1 is extensivelycleaved in postnatal kidneys, both in the CD and PT cells, atP3–P14 of WT mice. In the Pkd1V/V CD, the cystic change occursin the presence of PC1V but in the absence of any cleaved PC1products. Therefore, although uFL-PC1 appears sufficient forthe embryonic development of the CD, it is no longer enough forits postnatal maturation and maintenance, and the cleaved PC1molecules are required instead. Consistent with this notion,Pkd1V/� heterozygotes, which express slightly more uFL-PC1 butless cleaved PC1 products as compared with WT mice, appearnormal. However, PC1V is sufficient to protect the PT from

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Fig. 4. Distal nephron segment origin of cysts in Pkd1V/V kidneys. (A–H)Pkd1V/V kidney sections of various postnatal stages were double-stained withsegment-specific markers as indicated under each section. Lectin Lotus tet-ragonolobus (LTL), proximal tubule (PT); lectin Dolichos biflorus (DBA), col-lecting duct (CD) (26); AQP2, CD (27); Tamm–Horsfall protein (THP), both thethick ascending limb (TAL) and the distal convoluted tubule (DCT) (28); andNa-K-Cl Cotransporter 2 (NKCC2), TAL only (29). (I) Summary of cyst origin inPkd1V/V kidneys. Cysts are derived from DCT and CD (in red) (except the papillatip), and PT and TAL are not dilated.

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cystic dilation, indicating that uFL-PC1 is fully sufficient for thenormal structure of PT and the cleaved products are notessential.

There are several possible explanations for the apparentnonessential role of cleavage in the PT and possibly in the papillatip: (i) the cleaved products may be functional but redundant toother PC1 family members; (ii) the cleavage products may beinactive by-products of a default cis-autoproteolysis, and cleav-age could serve to keep the active uFL-PC1 at a low level,possibly because a high level is detrimental in proximal nephronsegments and the papilla tip; and (iii) unknown GPS-independent cleavage(s) not detected in our study could occur torelease active fragments that compensate the loss of GPScleavage products in Pkd1V/V PT. Further studies are required toresolve this issue.

Our finding of ubiquitous cleavage of endogenous PC1 is atodds with the results of most reports (19–21) using anti-PC1antibodies directed to the CTF. These antibodies detectedhigh-molecular-mass bands (�400 kDa) that were interpreted asbeing PC1, but did not recognize the far more abundant CTFband on the same Western blots. The possibility of a nonspecificdetection by these reagents should be considered.

In conclusion, our results support the hypothesis that uFL-PC1and GPS-cleaved PC1 products exert differential functions invivo, with uFL-PC1 playing a more critical role during embryonicdevelopment, and the cleaved PC1 products being essential for

distal nephron segments during the postnatal development of themouse kidney and the common bile duct and biliary tracts.Intracellular locations and functions of each PC1 moleculesremain to be determined.

Materials and MethodsGeneration of Pkd1V/V Knockin Mice. The targeting vector (Fig. 1 Ai) is made by using genomic DNA fragments derived from 129Svstrain. The T3041V mutation was introduced by using theQuikChange kit (Stratagene). Electroporation, selection, andscreening of ES cells (129/S6, CMTI-1) were carried out by usingstandard procedures. One clone (G8) with correct targeting wasidentified by using Southern blot analysis and long-rangegenomic PCR and was injected into C57Bl6 blastocysts togenerate chimeric mice that produced 129S6/C57Bl6 offspringcarrying the targeted Pkd1NeoV allele. The F1 Pkd1NeoV/� micewere bred with FLP transgenic mice to delete Neo. The resultingPkd1V/� offspring was intercrossed to generate Pkd1V/V mice,which were backcrossed onto the C57Bl6 strain for more thanfive generations for our analyses.

Genotyping of Mice. Initial genotyping was performed by using athree-primer PCR-based strategy (Fig. 1C i) with two forwardprimers, F24 and F2, and one reverse primer R1. The FLPtransgene was identified by PCR amplification of a 399-bpproduct with use of primers YSQ-F2 and YSQ-R (Fig. 1C ii). For

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Fig. 5. Cleavage patterns of endogenous PC1 in WT and Pkd1V/V mice. Endogenous PC1 was immunoprecipitated by using anti-CC and detected on Westernblot by anti-CC. The protein size marker and the position of uFL-PC1 and CTF are indicated. (A) Schematic diagram of the domain organization of mouse PC1.LRR, Leucine-rich repeat; R1 and R2–16, PKD repeats; CLD, C-type lectin domain; REJ, receptor for egg jelly domain. The cleavage site HLˆT3041 within GPS andthe resulting NTF (3,040 aa) and CTF (1,253 aa) are shown. The epitope recognized by anti-CC or anti-CT (the equivalent antibody against human PC1), andanti-LRR used in this study is indicated by a black bar. (B) Western blots demonstrating the specificity of anti-CC. It detects both C-terminally FLAG-tagged uFLand CTF from lysate of HEK cells with stable expression of mouse full-length Pkd1 cDNA (mPC1-F) after IP with either anti-FLAG or anti-CC (Right). These signalswere not detected from untransfected HEK cells (data not shown). Note that this pattern is identical to that of exogenously expressed human recombinant(hPKD1-F) or endogenous PC1 of HEK cells (HEK). (C–I) Western blots showing PC1 cleavage pattern in MEFs of various genotypes as indicated (C) and whole E12embryos of various genotypes as indicated (D). Bracket indicates nonspecific bands that are also present in E12.5 and E14.5 Pkd1�/� embryos (Right). (E) WTembryos of various stages as indicated. (F) Postnatal kidneys of WT and Pkd1V/Vmice at different stages. * indicates an unknown PC1-specific band that occursonly in Pkd1V/V cystic kidneys. (G) Primary PT and CD cells isolated from P5 WT and Pkd1V/Vkidneys (Left) and conditionally immortalized CD cells isolated fromP14 WT and Pkd1V/Vkidneys (Right). Same results were obtained for primary PT and CD cells isolated from P10 WT and Pkd1V/Vkidneys, respectively (data notshown). The purity of the PT and CD cells is verified by using the segment-specific markers [AQP2 for CD; APN, aminopeptidase N, for PT (30)] on Western blotof total lysates, or DBA by immunofluorescence. (H) Various organs of P3 WT and Pkd1V/Vmice. B, brain; L, liver; K, kidney; Lu, lung; H, heart. (I) Organs of WTmice at P19. S, spleen; P, pancreas. Note the low expression level of PC1 in kidney. Arrowhead in G and H indicates the high-molecular-weight band in Pkd1V/V

cells and organs that probably represents a modified uFL form.

18692 � www.pnas.org�cgi�doi�10.1073�pnas.0708217104 Yu et al.

genotyping of offspring of subsequent Pkd1V/� matings, only F2and R1 primers were used. Details regarding primer sequenceand PCR conditions are available on request.

Reverse Transcription-PCR. First-strand cDNA was synthesized byusing SuperScript II (Invitrogen) from 100 ng of total RNA andthen used as template for PCR amplification of Pkd1 transcriptswith the primers F2 and R24 (Fig. 1D). The PCR products weresequenced by using HXB-F15 within exon 25 (Fig. 1E).

Histology and Immunohistochemistry. Kidney and other specimensfrom different stages of animals were collected, fixed, embeddedin paraffin, and sectioned at 5 �m thickness for histologicalanalysis as described in ref. 6. H&E and MT stains were appliedby using standard protocols. Sections were first microwaved for10 min in citrate buffer solution (pH 6.0) to enhance antigenretrieval and then subjected to immunohistochemistry studies asdescribed in ref. 6. Indirect f luorescence microscopy was per-formed by using a Nikon Eclipse E600, and images werecaptured by using a SPOT-RT monochromic camera.

Cultured Cells. Conditionally immortalized MEFs were isolatedfrom E12 embryos containing a thermolabile SV40 tAg trans-gene (25) by using standard procedures. Cells were maintainedat 33°C with IFN-� and were moved to 37°C without IFN-� for3 days before harvest for analysis. PT and CD cells wereaffinity-purified from postnatal kidneys by using LTL andDBA-conjugated Dynabeads (Invitrogen), respectively. In brief,the kidneys were minced into small pieces and digested inMEM/F12 containing 0.2% collagenase, 0.2% hyaluronidase,and 0.001% DNase I at 37°C for 2 h with gentle agitation. Thedigested tissue was incubated with Dynabeads at 4°C for 30 min.

The cells bound to the beads were collected either directly forcell lysis (12) or, in the case of conditional immortalization, wereresuspended in culture medium for propagation.

Anti-PC1 Antibodies. To generate the polyclonal CC antibody, acDNA fragment corresponding to amino acid residues 4123–4291 of mouse PC1 was cloned into pET28c (Novagen). Afterexpression in Escherichia coli, the protein was affinity-purifiedwith Ni-agarose beads and then used to immunize rabbits andchickens. Chicken IgY antibody was purified by using theEGGstract IgY Purification System (Promega). Typically, puri-fied chicken anti-CC was used for IP and rabbit anti-CC was usedfor Western blot detection. Anti-CT and anti-LRR have beendescribed (12).

IP and Western Blot Analysis. Tissue samples taken from killedmice were immediately homogenized in lysis buffer (12) with aPolytron homogenizer (Kinematica). The homogenate was in-cubated for 1 h on ice and cleared of debris by centrifugation at17,000 � g for 10 min at 4°C. Ten milligrams of protein in 1 mlwas typically used for IP and Western blot analysis as describedin ref. 12.

We thank Dr. Knepper for providing anti-AQP2 and anti-NKCC2, Dr.Hoyer for anti-THP, and Dr. Hubbard (Johns Hopkins UniversityDigestive Diseases Basic Research Development Center) for anti-APN.We thank Jie Deng for technical assistance, and Drs. Alessandra Bolettaand Erum Hartung for discussions. This work was supported by NationalInstitutes of Health Grants DK062199 (to F.Q.) and DK48006 andDK51259 (to G.G.G.), a National Kidney Foundation grant (to L.F.M.),DC06471, ALSAC, and CA21765 grants (to J.Z.). G.G.G. is the IrvingBlum Scholar of the Johns Hopkins University School of Medicine.

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