BASIC RESEARCH www.jasn.org

Loss of the Ciliary Nek8 Causes Left-Right Asymmetry Defects

†‡ †‡ Danielle K. Manning,* Mikhail Sergeev, Roy G. van Heesbeen, Michael D. Wong,§ | †‡ Jin-Hee Oh, Yan Liu,¶ R. Mark Henkelman,§ Iain Drummond,¶ Jagesh V. Shah, and David R. Beier*

Divisions of *Genetics and ‡Renal Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; †Department of Systems Biology, Harvard Medical School, Boston, Massachusetts; §Mouse Imaging Centre (MICe), Hospital for Sick Children, Toronto, Ontario; |Department of Pediatrics, The Catholic University of Korea, Seoul, Korea; and ¶Nephrology Division, Massachusetts General Hospital, Charlestown, Massachusetts

ABSTRACT AmissensemutationinmouseNek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomi- zation of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function 2/2 2 2 . Notably, NEK8 and polycystin-2 (PC2) interact, and we found that Nek8 and Pkd2 / embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.

J Am Soc Nephrol 24: 100–112, 2013. doi: 10.1681/ASN.2012050490

Cystic diseases of the kidney are prevalent in the We previously reported that the mouse juvenile human population and there is compelling interest cystic kidneys (jck) model of recessive polycystic in understanding the functions of the associated kidney disease (PKD) is due to a missense mutation genes. The most common human cystic disease is in the Nek8 ; jck animals develop cysts in distal autosomal dominant polycystic kidney disease nephron segments and collecting ducts within the (ADPKD) with an incidence of 1 in 750 individu- first week of life and the disease rapidly progresses, als.1 ADPKDis caused by mutations in either PKD1 causing renal failure and death by 6 months of or PKD2, which encode the polycystin proteins PC1 age.11 Nek8 encodes a highly conserved member and PC2, respectively. PC1 is a G-protein coupled receptor that partners with PC2, a transient recep- tor potential polycystic family ion channel that reg- Received May 16, 2012. Accepted October 18, 2012. ulates both calcium entry into and intracellular Published online ahead of print. Publication date available at 2–7 calcium release within renal epithelial cells. www.jasn.org. The nephronophthisis (NPHP) types 1–11 and Correspondence: Dr. David R. Beier, Division of Genetics, NPHPL1 comprise a less common group of auto- Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, NRB somal recessive disorders, but they are collectively Room 458H, Boston, MA 02115. Email: [email protected]. the leading genetic cause of ESRD within the first harvard.edu – 3 decades of life.8 10 Copyright © 2013 by the American Society of Nephrology

100 ISSN : 1046-6673/2401-100 J Am Soc Nephrol 24: 100–112, 2013 www.jasn.org BASIC RESEARCH of the Nek family of serine/threonine , characterized determinants are expressed in the early embryo. The Nodal by an N-terminal kinase domain homologous to that of the gene encodes a ligand of the TGF-b family and is expressed NIMA protein that controls entry first in the node and subsequently in the left lateral plate me- during .12 Mammalian Neks have divergent C termini, soderm (LPM), and the transcription factor Pitx2 is later ex- and the NEK8 C terminus contains multiple repeats that are pressed in the left LPM; mutant alleles of each of these genes are homologous to RCC1, a guanine nucleotide exchange factor embryonic lethal with laterality defects.29–32 for Ran that is required for condensation,13–15 A majority of proteins associated with human cystic although NEK8 guanine nucleotide exchange factor activity diseases, including the polycystins and nephrocystins, localize has not been demonstrated. The jck mutation in Nek8 results to either the and/or the basal bodyof primarycilia.8,33 in a glycine to valine change in the second RCC repeat of the Similarly, NEK8 localizes to the base of renal cilia and is thus protein. Subsequent studies have reported missense mutations far the only kinase that has been localized to the ciliary axo- in the RCC domain-encoding regions of both rat Nek8 in the neme.34,35 Cilia defects are indeed present in jck cystic renal Lewis PKD model16 and human NEK8, in which three inde- epithelia and cells cultured from affected kidneys; mutant cilia pendent mutations were identified from 700 NPHP patients, are elongated and exhibit enhanced ciliary localization of PC1 making it a candidate for NPHP type 9.11,17 Together, these and PC2 compared with wild-type cilia.36,37 In addition, PC2 data demonstrate that the NEK8 C terminus is critical for its is hyperphosphorylated in jck kidneys and both wild-type and function and the protein plays an important role in the main- mutant NEK8 coimmunoprecipitate with PC2 from kidney tenance of renal tubule integrity in the postnatal kidney. Of lysates, suggesting that these proteins perform critical coop- note, all of the reported mutations of Nek8 are missense mu- erative functions.37 tations. We describe here the generation of a mouse carrying We originally hypothesized that the jck allele is hypomor- a null mutation of Nek8; in this line, mutant pups die shortly phic because the only overt phenotype in homozygotes is PKD, after birth and exhibit a variety of laterality defects and asso- mutant protein is expressed in the kidneys, and morpholino- ciated cardiac anomalies. mediated reduction of Nek8 in zebrafish causes pronephric Mutations in several PKD-associated genes cause laterality cysts.11 Here, we characterize the Nek8-null mutant and deter- defects in the developing mouse, and dysfunction of primary mine that ciliogenesis is intact, but left-right patterning is per- cilia is the critical link between the phenotypes. Kidney tubular turbed. We revisit morpholino knockdown in zebrafish and epithelial cells each express a primary that collectively demonstrate that Nek8 depletion causes laterality defects, act as mechanosensors of fluid flow through the tubules, confirming a conserved role for Nek8 in left-right patterning. transducing calcium-dependent signals hypothesized to keep Furthermore, we generated jck/Nek82 compound heterozy- the cells in a differentiated state.7 Early in development, the gotes; surprisingly, renal cystic disease in these mutants is establishment of appropriate asymmetry in mammals requires much less severe than in jck homozygotes, suggesting the jck the functions of two populations of cilia within the node, a pit allele of Nek8 encodes a gain-of-function protein. Finally, there in the ventral surface of the embryo. Motile cilia rotate and is marked similarity of Nek82/2 and Pkd22/2 embryonic generate a leftward fluid flow, and primary cilia subsequently phenotypes and we determine that PC2-associated activity is produce a left-sided calcium gradient.18,19 Mutations in Pkd2, abrogated in cells lacking NEK8. Kif3A, and Tg737(Ift88) illustrate how cilia are critical signal- ing centers in the kidney and the node. Loss of Pkd2 from the adult kidney results in PKD, whereas Pkd2-null embryos ex- RESULTS hibit left-right defects.20,21 Functionally, the PC1/PC2 com- plex mediates calcium influx in response to flow-induced Generation of Nek8-Null Mouse Model sheer stress in cultured renal cells, and loss of either protein To generate a Nek8-null allele, we obtained a C57BL/6 (B6) bac- abolishes the response22; similarly, Pkd2-null embryos do not terial artificial chromosome containing the Nek8 genomic locus. exhibit a left-sided calcium signal.19 Heterotaxy also occurs Nek8 exon 3 was targeted by recombineering using a cassette with loss of the Pkd1-related gene, Pkd1l1; the gene product composed of an internal ribosomal entry sequence (IRES), the localizes to cilia and interacts with PC2, forming a node-specific lacZ gene, and the neomycin resistance gene flanked by FLP complex required for proper asymmetry.23,24 recombinase recognition sites (Figure 1A). Nek8:lacZ was de- Alternatively, mutations that disrupt cilia assembly and/or signed to encode a truncated protein lacking the kinase domain structure cause both phenomena. PKD arises in the kidney- and a b-galactosidase reporter to recapitulate endogenous specific deletion of Kif3A, which encodes a kinesin subunit NEK8 expression. Nek8:lacZ-positive B6/129 hybrid embryonic required for ciliogenesis, and in Tg737 (Ift88/polaris) hypo- stem cell38 clones were injected into B6 blastocysts, and the re- morphs that exhibit shorter cilia with bulbed tips.25,26 Later- sulting chimeras were bred to obtain germline transmission of ality defects occur upon targeted disruption of Kif3A, Kif3B, the Nek8:lacZ allele. Heterozygous Nek8:lacZ carriers are viable or Tg737 (Ift88); each of these knockouts lack nodal cilia and and fertile with no obvious phenotypes, whereas Nek8:lacZ ho- die mid-gestation with randomized heart looping.18,27,28 mozygous pups die shortly after birth; carriers and homozygotes Downstream of flow-induced calcium signals, left-sided are obtained at expected 50% and 25% frequencies, respectively.

J Am Soc Nephrol 24: 100–112, 2013 Nek8 Disruption Causes Laterality and Renal Defects 101 BASIC RESEARCH www.jasn.org

(RPI), a “duplication” of the four-lobed right lung on the left side (Figure 2I); one-third of these mutants have normal ab- dominal situs, whereas two-thirds have inverted abdominal situs (Table 1). Mutants with laterality defects often have major structural cardiac anomalies.29,39–41 Therefore, magnetic resonance im- aging (MRI) and histologic analyses were performed to assess the hearts of Nek82/2 embryos. In three mutants with RPI we identified double outlet right ventricle (DORV), a defect in which both the pulmonary artery and aorta exit the right ventricle of the heart, accompanied by atrial and ventricular septal defects (A/VSD) (Figure 2, J and K and data not shown). DORV is best exemplified in lateral MRI images: the pulmo- Figure 1. Targeted disruption of Nek8 results in a true null allele. nary artery and aorta are captured in separate images in a (A) Nek8 exon 3 is disrupted with an IRES-lacZ:neomycin cas- fl wild-type embryo, but the structures run parallel to each sette. FLP recombinase recognition sequences (frt) ank neo- 2 2 mycin. (B) PCR genotyping distinguishes wild-type (+/+) embryos other in the same image in the Nek8 / mice. Although from heterozygous (+/2) and homozygous (2/2) lacZ-positive mice with situsinversustotaliscan be viable, as in inversin embryos. The wild-type band is amplified with F and R1 primers mutant mice,42 no Nek82/2 mice survive the perinatal pe- flanking exon 3. The mutant product is amplified with the F riod and we found A/VSD in mutants with complete situs primer and R2 primer from the lacZ sequence. (C) RT-PCR analysis inversus (Figure 2, L and M). confirms that the lacZ reporter is spliced out of the mutant allele. To further examine the requirement for Nek8 in establish- rtF in exon 2 and rtR in exon 4 amplify a 400 bp product and ing normal patterning in a vertebrate system, we utilized mor- a 150 bp product from wild-type and Nek8 mutant MEF cDNA, pholino knockdown of Nek8 in zebrafish. We previously used respectively. (D) No NEK8 is detected via Western blot analysis of this approach to evaluate cystogenesis in adult fish,11 but lat- MEF lysates. erality was not assessed. In this analysis, randomization of heart looping occurs in a dose-dependent manner in zebrafish embryos injected with a morpholino targeted to the initiator Heterozygous- and homozygous-Nek8:lacZ embryos were methionine codon of Nek8 (P,0.001; Figure 2N). analyzed for b-galactosidase activity, but activity was not detected in PCR-positive samples (Figure 1B and data not Early Laterality Markers Are Misexpressed in Nek82/2 shown). We derived mouse embryo fibroblasts (MEFs) from Embryos but Nodal Cilia Are Intact E14.5 wild-type and homozygous embryos, analyzed the Left-right asymmetry mutants have defects in nodal signaling cDNA from the cells, and discovered the targeted exon 3 is that are assessed via in situ hybridization of Nodal and Pitx2 spliced out of the mutant transcript, rendering the reporter between E8.0 and E8.5.43–45 In wild-type presomite embryos, ineffective (Figure 1C and data not shown). However, the ab- Nodal expression is symmetrical in the node. By the 3–4somite errant splicing of exons 2–4 causes a frameshift and a creates stage, it becomes asymmetrically expressed in the node, staining premature stop codon resulting in a true null allele of Nek8, stronger on the left side than the right, and expression is re- which we herein refer to as Nek82, because no protein is stricted to the left LPM (Figure 3A). In contrast, in Nek82/2 detected in homozygotes by Western blot analysis (Figure 1D). embryos at the 3–4 somite stage, Nodal expression within the nodes was always symmetrical (n=9) and was absent from the Nek82/2 Embryos Exhibit Randomization of Left- LPM in seven of nine embryos, bilateral in one embryo, and Right Asymmetry and Cardiac Defects expressed in the left LPM in one embryo (Figure 3, B–D and Nek82/2 mice die shortly after birth and mid-gestation mu- Supplemental Figure 1). We examined embryos at the 6–8 so- tants frequently exhibit edema and focal hemorrhages that are mite stage for Pitx2 expression, which is normally apparent in suggestive of cardiac defects (Figure 2, A–D). Upon autopsy, the headfolds and a discrete region of the left LPM of wild-type we observe randomization of left-right asymmetry in the mu- embryos (Figure 3E). Pitx2 was appropriately expressed in the tants. The characteristic asymmetry of mouse body plan is headfolds of stage-matched Nek82/2 embryos (n=6), but was defined in the thorax by a four-lobed right lung and single absent from the LPM in four of six mutants (Figure 3F), right- left lung lobe with a left-oriented heart apex; in the abdomen, sided in one mutant, and correctly expressed in one mutant. the stomach and adjacent spleen reside on the left side (Figure Several laterality mutants have short nodal cilia or lack them 2, E–G). Approximately 15% of Nek82/2 mutants have nor- altogether, which disrupts asymmetric signaling downstream mal body asymmetry, whereas 24% display situs inversus of nodal flow.18,27,28 Because NEK8 localizes to renal epithelial totalis, in which the body plan is a mirror image to that of a cilia, we examined the nodal cilia of E8.0 embryos using im- normal fetus (Figure 2, E, F, and H). However, a majority of munofluorescent detection of the ciliary marker acetylated Nek82/2 mutants (61%) exhibit right pulmonary isomerism a-tubulin and the anti-NEK8 antibody. Wild-type and

102 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 100–112, 2013 www.jasn.org BASIC RESEARCH

Nek82/2 nodes are indistinguishable (Figure 3, G and H) and NEK8 is expressed in the nodal cilia of wild-type E8.0 embryos (Figure 3I). Taken together, the in situ hybridization and im- munofluorescence data suggest that Nek8 performs a critical function in laterality determination, but is not directly re- quired for ciliogenesis.

Nek82/2 Kidneys Do Not Develop PKD Nek82/2 pups die within hours of birth but they can produce urine, indicating that the kidney is functional. We examined renal development in Nek82/2 embryos and the mutant kid- neys develop similarly to those of wild-type through E15.5; however, by E16.5 and through P0, glomerular cysts are ap- parent in periodic acid–Schiff (PAS)-stained samples (data not shown and Figure 4, A and B). In addition, P0 Nek82/2 proximal tubules appear thin-walled and slightly dilated compared with the wild-type proximal tubules, which are thicker due to intact brush borders on the apical surfaces. To further assess the tubular phenotype, we stained kidneys with the fluorescein-conjugated Lotus tetragonolobus lectin (LTL) and Dolichos biflorus agglutinin (DBA) to identify the proximal tubules and collecting ducts, respectively. LTL staining confirmed that the Nek82/2 proximal tubules are indeed dilated at P0 but not at E18.5, whereas the DBA stain- ing showed that P0 mutant collecting ducts are largely un- affected (Figure 4, C–F and data not shown). Importantly, the abundance and appearance of cilia in wild-type and Nek82/2 tubules is similar (Figure 4, C–F, acetylated a-tu- bulininred). Although ciliogenesis appears normal in Nek8 mutant nodes and kidneys, it is difficult to identify overt length or structural defects via immunofluorescence in these tissues. Therefore, we analyzed cilia in control and Nek8-deficient mouse inner medullary collecting duct (IMCD) cells (Figure 4, G and H) as well as in wild-type and null MEFs (Figure 4, JandK).TogenerateNek8-depleted IMCD cells, small hair- pin RNA (shRNA) against Nek8 was introduced and indi- vidual clones were isolated and expanded; NEK8 is not detected via WesternblotinlineN8KD10comparedwith the vector control clonal (pLKO) line (Figure 4I). There are no morphologic or length differences between the cilia of wild- type and Nek8-deficient cells in culture: pLKO and N8KD10 Figure 2. Nek82/2 mice exhibit laterality and cardiac defects. (A–D) Lateral views of littermates. E14.5 wild-type embryo (A) compared with Nek82/2 embryo (B) with edema (arrows) and focal hemorrhages. E16.5 wild-type embryo (C) is larger than the defects. (J and K) MRI images through thoraces of a wild-type and mutant (D). (E–H) E18.5 wild-type compared with a mutant with a null littermate with RPI. Sagittal views show that wild-type pul- SI. (E) Lateral views of a wild-type embryo (left) and a null em- monary artery (J) and aorta (J’) are present in distinct images. (K) bryos (right) illustrate abdominal situs defect in the mutant. (F) Mutant pulmonary artery and aorta run parallel in the same image. Frontal views of the embryos in E, in which internal organs mirror (L and M) H&E-stained transverse cardiac sections. (L) The wild- each other. (G and H) Hearts and lung lobes dissected out of the type heart has intact atrial (*) and ventricular septa. (M) The SI thoracic cavities of embryos in E and F. (G) Wild-type lungs mutant heart exhibits atrial (*) and ventricular (arrow) septal de- contain four right lobes and one left lobe (R4, L1). (H) The mutant fects. (N) Cardiac looping occurs in Nek8-ATG MO zebrafish em- with SI has one right lobe and four left lobes (R1, L4). (I) Heart and bryos in a dose-dependent manner. Scale bars, 1.0 mm in A–I; 100 lungs dissected out of a mutant with RPI; right and left lungs are mminJ–M. SI, situs inversus;s,stomach;sp,spleen;R,right;L,left; four-lobed (R4, L4). (J–M) Nek82/2 embryos exhibit cardiac h, heart; pa, pulmonary artery; a, aorta; vs, ventricular septa.

J Am Soc Nephrol 24: 100–112, 2013 Nek8 Disruption Causes Laterality and Renal Defects 103 BASIC RESEARCH www.jasn.org

Table 1. Summary of laterality defects in Nek82/2 embryos Because PC2 expression/localization is intact in Nek8-null Thoracic Situs tissues, we hypothesized that PC2 activity may be perturbed Abdominal Situs fi SS RPI SI in NEK8-de cient cells. To investigate PC2 function, we subjectedculturedwild-typeandN8KD10(knockdown) SS 20 (39) 8 (15) 12 (24) 0 IMCD cells to fluid shear stress and measured changes in SI 31 (61) 0 19 (37) 12 (24) 2+ fl Total 51 (100) 8 (15) 31 (61) 12 (24) PC2-dependent intracellular Ca in ux across the membrane Data are shown as the number of embryos (% of total). SS, situs solitus;SI, using live cell microscopy; PC2 is properly localized in wild- situs inversus. type and N8KD10 cilia under the conditions used in the assay (Supplemental Figure 2). The cells stably express a genetically cilia lengths are 3.361 mmand3.060.6 mm, respectively; encoded calcium ion indicator, GCaMP3, used to monitor wild-type and Nek82/2 MEF cilia lengths are 2.960.7 and changes in intracellular Ca2+ levels.48,49 Live fluorescence im- 2.860.9 mm, respectively. Statistical analysis using a nonpara- age time series were acquired while Ca2+ depleted cells were metric ANOVA test reveals that these differences are not sig- exposed to fluid shear stress. As expected, wild-type IMCD cells nificant, suggesting that ciliogenesis can occur normally in the exhibit a robust response to fluid shear stress (Figure 6, D and absence of NEK8. G). PC2-depleted IMCD cells50 were used as a negative control and show significantly reduced fluid flow response (Figure 6, E jck/Nek82 Compound Heterozygotes Have Less and G). The N8KD10 cells also have a reduced response to flow, Severe Cystic Disease Than jck Homozygotes suggesting that PC2 function may be perturbed in the absence We hypothesized that the jck allele of Nek8 is hypomorphic, of Nek8 (Figure 6, F and G). and to test this we intercrossed jck/1 and Nek81/2 mice to generate compound heterozygous jck/Nek82 animals. Wild- type, jck/+, jck/Nek82,andjck/jck mice were sacrificed at 7 DISCUSSION weeks of age, the kidneys were weighed and histology was performed to assess disease severity. Surprisingly, jck/Nek82 The Nek8 gene, which is mutated in the jck mouse model of animals develop very mild renal cystic disease and the com- PKD, encodes the only kinase that to date has been localized to bined kidney weights are not much greater than those of the ciliary axoneme. In this study we report that its targeted wild-type mice (Figure 5, A–D). disruption in the mouse is lethal and causes laterality defects, Because Nek82/2 pups die at birth, we analyzed the cys- further demonstrating a link between cystogenesis and left- togenic potential of null, jck, and compound mutant embry- right asymmetry. Importantly, there is a conserved require- onic kidneys in an explant culture assay.46,47 Kidneys were ment for the gene in the specification of vertebrate left-right removed from E14.5 embryos and cultured for 5 days with asymmetry as knockdown of Nek8 expression in zebrafish or without a cyst-inducing cAMP analog. In the presence of causes randomization of heart looping, a result described 8-Bromo-cAMP, jck/jck kidneys develop the greatest numbers both here and by an independent group.51 of cysts, whereas Nek82/2 kidneys have the least cystogenic A small number of genes implicated in human renal cystic potential of all the genotypes (Figure 5, E and F). Taken to- diseases cause both PKD and laterality defects when disrupted gether, the findings that jck/Nek82 adult and cultured embry- in the mouse. Three such examples include Pkd2, inversin,and onic kidneys develop less severe cystic disease than homozygous Nphp3; PKD2 is mutated is 15% of human ADPKD cases and jck kidneys, and that the Nek8-null kidney explants do not mutations in INVS (inversin) and NPHP3 cause NPHP types develop tubular cysts, suggest the jck mutation results in a 2 and 3, respectively.1,52,53 NEK8 has been identified as an gain-of-function protein. interacting partner of each of the mouse protein counterparts: NEK8 and PC2 reciprocally coimmunoprecipitate from kid- Nek82/2 Phenotypes Resemble Those Caused by Loss ney and cell lysates,37 and NEK8 has been shown to colocalize of PC2 Function with inversin and NHPH3 in the base of the cilium in an The Nek82/2 phenotypes are remarkably similar to those of inversin-dependent manner.35 The association of NEK8 with Pkd22/2 embryos, including randomization of left-right each of these proteins is intriguing due to the similarities asymmetry, RPI, cardiac malformations, and glomerular cysts and some of the differences we identified in the jck and in late-gestation kidneys,21,39 and NEK8 and PC2 have been Nek8-null mouse models compared with Pkd2, inversin,and shown to interact.37 Therefore, we sought to determine Nphp3 mutants. whether the Nek82/2 defects are due to loss of PC2 expres- With regard to left-right asymmetry, Pkd22/2 embryos die sion or a change in localization. PC2 localizes to all node in utero and 90% of the mutants exhibit RPI,39,21,54 whereas a monocilia19 and we observe its localization is identical in majority of inversin mutants exhibit situs inversus.55–57 The E7.75 wild-type and Nek82/2 embryo nodes (Figure 6, A observation that Pkd22/2 early embryo nodes fail to elicit and B). In addition, we do not detect gross differences in an asymmetric calcium signal in response to flow19 likely ac- PC2 levels via Western blot between littermatched E18.5 counts for the absence of Nodal and Pitx2 expression in the wild-type and Nek82/2 kidney lysates (Figure 6C). LPM, causing the high percentage of RPI in mutant embryos.

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mutants are more similar to Pkd22/2 than to the inversin mutant mice. We de- termined that Nek8-null phenotypes are not due to loss of PC2, because the protein is expressed and properly localized in mu- tant embryos, tissues, and cells. Therefore, we utilized a fluid flow assay to measure PC2-dependent mechanosensitive activity in cells lacking NEK8. IMCD cells stably knocked down for Pkd2 or Nek8 exhibit de- creased calcium influx in response to shear stress compared with wild-type cells, sug- gesting PC2 is not fully functional in the absenceofNEK8.GiventhatPC2isre- quired for both the IMCD cell responses to flow and the calcium gradients detected in the mouse embryo node, we hypothesize that the ability of PC2 to “sense” nodal flow and elicit a left-sided signal is diminished in nodes lacking NEK8. Perhaps NEK8 is a critical component of the PC2 channel complex that is required to mediate the sig- naling processes downstream of nodal cal- cium flux that are indispensable for the establishment of appropriate left-right asymmetry. Similarly to other heterotaxy mutants, Nek82/2 embryos exhibit severe cardiac malformations that include DORV cou- pled with A/VSD.59 It is not surprising to find such abnormalities in animals with RPI, because aberrant heart development accompanies the inappropriate symmetry in the thoracic cavity. However, we identi- Figure 3. Laterality marker genes are misexpressed in Nek82/2 embryos but nodal fied cardiac septal defects in mutants with cilia are intact. (A–F) In situ hybridization of laterality markers confirms signaling de- complete situs inversus, a condition that is fects in Nek82/2 embryos (arrows indicate LPM). (A–D) Nodal is expressed in the not lethal if the inverted body plan is ap- node proper in 4–5 somite wild-type and null embryos, as well as in the wild-type left propriately established, suggesting that LPM shown in A. Mutant embryos lack LPM staining (B) or have bilateral (C) or left LPM Nek8 may play a role in cardiac morpho- expression (D). Pitx2 is expressed in 6 somite wild-type and null headfolds and in the genesis independent of the initial left-right wild-type left LPM (E), whereas mutant embryos usually lack LPM expression (F). Anti- asymmetry defect, as is the case for inversin acetylated tubulin immunofluorescence of E8.0 embryos shows that wild-type nodal cilia (G) (n=10) and null nodal cilia (n=5) (H) appear similar. High-magnification view of mutants. It was originally reported that wild-type nodal cilia (I) (arrows) stained with anti-acetylated tubulin (green) and anti- nearly 100% of inversin mutants display NEK8 (red); NEK8 localizes to all node monocilia (merge; inset, NEK8 at base of cilia) situs inversus totalis; therefore, the thoracic and is not detected in Nek82/2 nodal cilia (J). Scale bars, 200 mminA–I. R, right; L, situs was “normal reversed” compared left. with wild-type littermates. However, a later study of inversin homozygotes found that 90% of the pups did have situs inversus,but Interestingly, inversin nodal cilia produce a weak net leftward 10% were identified with situs solitus; furthermore, cardiac flow much slower than wild type,58 which may cause right- malformations including outflow tract and septal defects sided Nodal expression,44 more frequently lead to inversion of were found in 37% of the pups, including those few with the body plan, and preclude the development of RPI in the normal thoracic situs.60 Perhaps Nek8 and inversin are re- thorax. A majority of Nek8-null embryos exhibit RPI whereas quired in the cardiac cilia present in mid-gestation mouse only 24% have situs inversus, and both Nodal and Pitx2 are embryos,61 a biologic question best addressed by conditional frequently absent from the left LPM of mutants; thus, Nek82/2 deletion of the gene.

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Our original observations that PKD develops in jck homo- zygotes, affected animals express the mutant protein, and morpholino knockdown of zebrafish Nek8 causes pronepeph- ric cysts all suggested the jck allele represented a partial loss of function. This hypothesis was consistent with analysis of the Ift88 gene, which initially established the critical link between renal cysts and left-right asymmetry, because the hypomor- phic Tg737 allele causes PKD, whereas the null mutation cau- ses laterality defects.26,28 Similarly, the pcy mouse carries a missense mutation in the Nphp3 gene and develops slowly progressive PKD, whereas null mutants die mid-gestation with laterality and severe, complex cardiac defects.52,62 Of note, pcy/Nphp32 compound heterozygous mice are viable and develop severely cystic kidneys by week 12. This intermediate phenotype indicates that the pcy allele of the gene is hypomorphic compared with the null allele.62 We crossed the Nek8-null allele with jck carriers anticipating a similar result; that is, compound heterozygous mice would have a PKD phenotype more severe than that of jck homozy- gous mice. On the contrary, jck/Nek82 compound heterozy- gotes develop less severe PKD than the homozygous jck mutants. The null pups die shortly after birth and the kidneys do have glomerular cysts and dilated proximal tubules, but the defect is likely due to impaired glomerular function rather than early signs of NEK8-dependent PKD. Supporting this notion is that jck mutant glomeruli and proximal tubules do not develop cysts; it is the distal DBA-positive collecting ducts that are affected.36,37,63 Therefore, we utilized embryonic kidney explant culture to further explore the cystic potential of the mutant kidneys.46 This in vitro system recapitulates what we observe in the mice: Nek82/2 kidneys do not develop tubular cysts, whereas jck/jck homozygous kidneys are dramatically affected and jck/2 kidneys have an intermediate phenotype. The data therefore suggest that the jck allele encodes a gain- of-function protein, a hypothesis not initially considered be- cause jck/+ adults do not exhibit PKD. However, Natoli et al. performed explant cultures with kidneys harvested a day ear- lier than in our study, and observed a significant difference between wild-type and jck/+ kidneys, reporting cyst percen- Figure 4. Nek82/2 kidneys exhibit glomerular cysts and proxi- tages of 2.5% for wild-type and 7.5% for jck/+ compared with mal tubule dilation. (A and B) PAS-stained kidneys from P0 pups. 47 (A and A’) The wild-type kidney has normal glomeruli and prox- 15% for jck/jck kidneys. In the adult jck/+ kidney, perhaps imal tubules stain pale purple (arrows). (B and B’) The mutant the presence of wild-type NEK8 is protective against the del- kidney develops glomerular cysts and proximal tubules are dis- eterious effect of the mutant protein. It is currently unclear tended. FITC-conjugated LTL lectin marks proximal tubules (ar- how the jck allele may result in a gain-of-function protein. A rows) and confirms that the wild-type tubules are intact (C), recent study demonstrated that wild-type NEK8 and the jck- whereas the mutant tubules adjacent to glomerular cysts are equivalent NEK8G442V variant were similarly active in in vitro distended (D). (E and F) FITC-conjugated DBA lectin marks the kinase assays toward b-casein and histone, but it remains collecting ducts (arrows). Wild-type tubules (E) and mutant tu- unknown whether NEK8G442V exhibits altered protein bules (F) are similar in size and shape and cilia are present in both (red puncta, anti-acetylated tubulin immunofluorescence). Cilia (arrows; red, anti-acetylated tubulin) of control (pLKO) (G) and Nek8-knockdown (N8KD10) (H) IMCD cells are of similar mor- phology and length. (I) Western analysis of IMCD lysates illus- (J) and Nek82/2 MEFs (K) are of similar morphology and length. trates N8KD10 cells lack NEK8 (arrow) compared with the larger Scale bars, 100 mminA–F; 10 mminG,H,J,andK.Original nonspecific band recognized by the antibody. Cilia of wild-type magnification, 310 in A and B; 340 images of boxed region in A’ and B’. g, glomeruli; gc, glomerular cysts.

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is upregulated and mislocalized in jck cys- tic tissues.37 Similar to loss of Pkd2, its transgenic overexpression in the kidney causes cysts65; perhaps the excess PC2 in jck kidneys underlies the progressive cys- togenesis we observe in these mice. Over- all, our data provide in vivo support for the importance of the biochemical interaction identified between NEK8 and PC2 and compel additional studies to determine the nature of the relationship between these two proteins, both of which are crit- ical for the establishment of left-right asymmetry and are required later in life for the maintenance of renal epithelial in- tegrity.

CONCISE METHODS

Generation of the Nek8:lacZ Allele, Mouse Husbandry, and Genotyping EL350 E. coli were transformed with a C57BL/6 (B6) BAC (RP23–163A8) containing the Nek8 genomic locus and exon 3 was targeted with an IRES-lacZ cassette via gap repair.66 Nek8:lacZ was electroporated into B6/129 hybrid embry- onic stem cells38,67 and a positive clone was in- ’ Figure 5. jck/Nek8-compound heterozygotes exhibit moderate PKD and Nek82/2 jected at the Brigham and Women sHospital kidneys do not develop cysts. (A–C) H&E stained kidneys at 7 weeks. (A) jck/+ kidneys Transgenic Core. Chimeric males were crossed do not develop PKD. jck/Nek82 kidneys (B) develop moderate PKD compared with to B6 females and germline transmission of the the severe PKD of jck/jck mutants (C). (D) Comparison of combined kidney weights Nek8:lacZ allele was confirmed by PCR. Male between wild-type (n=7), jck/+(n=8), jck/Nek82 (n=8), and jck/jck (n=15) animals. (E and female Nek8:lacZ carriers were intercrossed and F) Embryonic kidney explant culture cyst formation assay results. Nek82/2 kid- and homozygotes were not present at weaning. neys do not develop many cysts, jck/Nek82 kidneys develop moderate cysts, and jck/ Matingswereset,embryoswereharvestedat jck cystogenic potential is the greatest when cultured with 8-Br-cAMP. Error bars in E various gestational time points, and tails were – represent SEM. Scale bars, 0.5 mm in A CandF. used for genotyping to confirm homozygous offspring. A three-primer genotyping strategy was utilized as follows: a forward primer that interactions and/or substrate specificity in vivo that would anneals to both wild-type and mutant loci 59-GACACCAT- affect its function.64 Another potential consequence of the TAGGCGCCTTCC-39 was combined with a wild-type–specific mutation is the mislocalization of jck-specific NEK8 proteins; reverse primer 59-CTTCTCAAAGCAGGCCTTGG-39 and a mutant- both Smith et al. and Zalli et al. suggest the mutant NEK8 specific reverse primer 59-GGGGATCCATATTATCATCG-39. Mainte- fails to localize to cilia and centrioles in cells cultured from nance and genotyping for the jck mouselinehasbeendescribed jck/jck kidneys and RPE cells, respectively.36,64 However, previously,11 and intercrosses were performed between jck/+ females Sohara et al. demonstratethatinsectionsofintactjck and Nek8+/2 males to obtain compound heterozygotes. All animals homozygous kidneys, ciliary expression of the mutant pro- were housed in accordance with Harvard Medical School Animal tein is detected along the entire axoneme, and is not confined Resources and Comparative Medicine regulations. to the proximal region in which it is normally localized.37 Fur- ther studies are therefore required to understand the conse- Zebrafish Morpholino Injection and Phenotype quences of the jck missense mutation. Analyses The fact that jck homozygotes do not exhibit laterality For Nek8 morpholino knockdowns, wild-type strain TU-AB zebra- defects suggests that the missense mutation does not abro- fish embryos were injected at the one- to four-cell stage with 4.6 nl gate PC2 activity. However, the PKD phenotype of jck mice of a 0.125 mM, 0.25 mM, or 0.5mM solution of a Nek8 ATG trans- may still be a consequence of PC2 dysregulation, because PC2 lation blocking oligo: 59-CTTCTCATACTTCTCCATGTTTTCG-39.

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Cell Lines, RT-PCR, and Western Blot Analyses MEFs were derived from E14.5 embryos using standard procedures67 and subsequently im- mortalized via retroviral infection of the large Tantigen.68 Transcript analysis was performed on RNA isolated with Trizol (Invitrogen) from wild-type and mutant MEF lines; Superscript One-step RT-PCR (Invitrogen) was used to amplify transcripts with primers specifictoNek8 exon 2(59-CAACCACCCCAACGTCATCG-39)and exon 4 (59-GCTACTCTTCTGGTTGTAGGG-39). Nek8 knockdown was performed in IMCD cells50 by lentiviral infection of shRNA-express- ing constructs that were generated by annealing complementary primers and cloning into the pLKO-puromycin vector (shRNA target se- quences were obtained from the Broad Institute shRNA Consortium). A hairpin targeting nucle- otide 1570 (sh1570) conferred the most robust knockdown when analyzed in a single infection; we subsequently isolated clonal lines and screened for the loss of NEK8 expression by Western blot and identified line N8KD10. For fluid shear stress experiments, stable wild-type, N8KD10, and PC2-deficient IMCD cell lines expressing GCaMP3 were generated using a blasticidin-resistant retroviral vector as previously described.69 GCaMP3-positive cells were grown in MatTek chambers (35-mm petri dishes with a bottom coverslip insert) to 40%– 50% confluency before serum starvation (0.5% FBS) (MatTek, No. 1.5). Cells were serum starved for 96 hours before fluid shear stress experiments in order to reach their highest Figure 6. PC2 is expressed in Nek8-deficient embryos and cells but flow sensing is frequency of ciliation. perturbed. Whole-mount immunofluorescence of E7.75 embryos shows PC2 locali- For Western blot analyses, cell and tissue zation in both wild-type (A) and Nek82/2 nodal cilia (B). (C) Western blot analysis lysates were subjected to SDS-PAGE. Blots were shows that PC2 is expressed at similar levels in E18.5 wild-type and Nek82/2 kidney incubated with primary antibodies overnight at lysates. (D–G) Effects of fluid shear stress exposed to cultured IMCD cells on PC2- 4°C at the following concentrations: anti-NEK8 2+ dependant intracellular Ca influx. The representative time traces of GCaMP3 fluo- (1:500011), anti-PC2 (1:100070), and anti-tubulin fl rescence intensity (normalized by the initial uorescence measured before introducing (1:500; Sigma). the sheer stress to the cells) are shown for Ca2+-depleted wild-type PC2-knockdown (E) and NEK8-knockdown (N8KD10) IMCD cells (F). The live cell media contains 1.25 mM CaCl2 at the time of the flow stimulation. Error bars represent SEM. (G) The MRI maximum (peak) values of normalized GCaMP3 fluorescence intensity were obtained A multi-channel 7.0-T MRI scanner (Varian from multiple single time traces and averaged over the cell population. Error bars Inc.) with a 6-cm inner bore diameter insert represent the SD of multiple single cell measurements. Nonparametric t tests were gradient set was used to acquire anatomic im- performed on the selected data sets (***P,0.001, **P,0.01). All of the measurements ages of E18.5 mouse embryos. Before imaging, were carried out under identical conditions. Scale bars, 10 mminAandB;5mminA’ samples were immersed in 2 mM ProHance in and B’. PBS (gadoteridol; Bracco Diagnostics Inc.) for a week, and were then placed into 2 mM ProHance Heart looping was determined at 29 hours postfertilization. Un- in low-melting-point agarose (Fisher). Parameters used in the scans injected embryos or embryos injected with a scrambled control were optimized for contrast within the mature mouse embryo as morpholino 59-CCTCTTACCTCAGTTACAATTTATA-39 exhibi- follows: a T2-weighted, three-dimensional fast spin-echo sequence, ted normal development. with repetition time/echo time (TR/TE) of 325/30 milliseconds, eight

108 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 100–112, 2013 www.jasn.org BASIC RESEARCH averages, fieldofviewof14314325 mm, and matrix size of divided by the area of the kidneys, as determined using ImageJ soft- 34833483624, giving an image with 40 mm isotropic voxels. ware and data were analyzed with Prism software. The SEM was calculated from the cyst percentages of pooled data from at least Histology and Immunofluorescence four independent experiments (n=10 kidneys per genotype), and Embryos and kidneys were fixed in Bouin’s solution and were em- the P values were determined using the Bonferroni multiple compar- bedded in paraffin, and 6-mm sections were cut for hematoxylin and ison test. eosin (H&E) staining. Kidneys were fixed in 4% paraformaldehyde and were embedded in paraffin, and 8-mm sections were cut for PAS Microscopy and Imaging staining and immunofluorescence/lectin procedures. PAS staining Whole-mount embryos and renal explants were analyzed on a Leica

(Sigma) was performed according to the manufacturer’s instructions. DM125 dissecting microscope and images were captured with Leica For immunofluorescence and lectin staining, slides were subjected to FireCam software. Histologic and immunofluorescent experiments citrate buffer antigen retrieval and incubated with antiacetylated tu- were analyzed on a Zeiss Imager.Z1 microscope and images were bulin antibody (Sigma) at a 1:10,000 dilution combined with either captured with AxioVision software; immunofluorescence slides FITC-conjugated LTL or DBA (Vector Labs) at 4°C overnight. Goat were imaged with the ApoTome engaged for better resolution of cilia anti-mouse Alexa Fluor 594 (Invitrogen) was used to visualize the and nuclei. tubulin and slides were mounted with Vectashield (Vector Labs). Whole-mount immunofluorescence was performed on E7.75– Fluid Shear Stress Experiments and Statistical Analyses 71 E8.0 embryos with anti-NEK8 (1:500) or anti-PC2 (1:1000 )anti- GCaMP3-positive IMCD cells were introduced to CO2 independent bodies overnight at 4°C. Embryos were then incubated with antia- Hanks balanced salt solution media supplemented with HEPES (25 cetylated tubulin for 1 hour at room temperature followed by goat mM final concentration), nonessential amino acids, sodium pyru- anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 647 secondary vate, glucose and GlutaMAX. Intracellular calcium stores were antibodies (Invitrogen). Embryos were mounted under individual purged by challenging cells with 2 mM EGTA and 100 mMATP coverslips with Vectashield (Vector Labs) and processed for genotyp- before Ca2+ influx measurements. Cells were introduced back to ing after imaging. EGTA-free Hanks balanced salt solution–based media and the cell For cilia length analysis, IMCD cells and MEFs were plated on chambers were mounted on the stage of a Nikon Eclipse Ti inver- glass coverslips (VWR Scientific); at approximately 30% conflu- tedmicroscopeequippedwithaNikonPlanApo203A0.75NA ence, the cells were serum-deprived for 48 hours before incubation objective lens and a CoolSnap-HQ (Photometrics). The Nikon with antiacetylated tubulin and Alexa Fluor 647. A nonparametric FITC cube was used to efficiently reflect 488 nm wavelength and ANOVA test was carried out to compare the cilium length distribu- pass the emission wavelengths into the charge-coupled device cam- tions obtained. era detection channel. While fluorescence image time series were acquired, cells were challenged with fluid shear stress via the con- 2+ Whole-Mount In Situ Hybridization and Fixed Tissue trolled addition of Ca media. The acquisition settings were kept Genotyping constant for all samples so that valid comparisons could be made E8.0–E8.5 embryos were harvested and fixed overnight in 4% para- between measurements from different data sets. Acquisition param- formaldehyde at 4°C, and were then dehydrated through a graded eters were set within the linear range of the charge-coupled device methanol series. For hybridization, embryos were rehydrated and camera detection. taken through the whole-mount in situ procedure described by A custom MATLAB (The MathWorks Inc) subroutine was written Wilkinson.72 Embryos were hybridized with digoxigenin-labeled to analyze acquired image time series. The mean intensity of the mPitx2c73 and nodal44 probes overnight at 65°C, incubated with anti- background noise was calculated from empty dark regions in the digoxigenin antibody (Roche) conjugated with alkaline phosphatase images and each frame was individually corrected. Regions of interest and probes were visualized with BM purple substrate (Roche). Whole (ROIs) from individual cells were chosen for the subsequent analysis. litters were processed simultaneously; after images were obtained, The mean intensity values measured from the first 30 frames (before DNA was prepared from the embryos for genotyping. The forward exposing cells to fluid shear stress) of acquired image time series primer is the same as described above, whereas the reverse primers defined ROIs and used to normalize the values of fluorescence wild-type 59-CAGAGCCAGCAGGATCTGCAC-39 and mutant 59- intensity to unity. The SDs of the recovered mean values were obtained CGGCTTCGGCCAGTAACGTTAG-39 efficiently amplify DNA tar- from the analysis of multiple ROIs in multiple independent experi- get sequences from fixed tissue. ments.

Renal Explant Culture Renal explant culture was performed as described by Natoli et al.47 ACKNOWLEDGMENTS Kidneys were harvested from E14.5 embryos and transferred to Corning transwell filters suspended in DMEM/F12/10% FBS. At 24 We thank A. Kumar and M. Keuhn for the nodal in situ hybridization hours, media were supplemented with either DMSO or 100 mM8- probe and N. Kurpios and C. Tabin for the mPitx2c in situ hybrid- bromo-cAMP (Sigma) and changed daily for 4 days. Cyst percentage ization probe. PC2 antibodies were provided by G. Pazour and T. was calculated from images by obtaining the total area of the cysts Benjamin. We thank R. Bronson at the Harvard Medical School

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Rodent Histopathology Core for analysis of cardiac serial sections and in the Lewis polycystic kidney rat localises to a conserved RCC1 domain W. Pu for MRI assistance. in Nek8. BMC Genomics 13: 393, 2012 Support for this work was provided by grants from the National 17. Otto EA, Trapp ML, Schultheiss UT, Helou J, Quarmby LM, Hildebrandt F: NEK8 mutations affect ciliary and centrosomal locali- Institutes of Health to J.V.S. (NIH P50 DK074030 and Genzyme zation and may cause nephronophthisis. JAmSocNephrol19: 587– GRIP), I.D. (NIH R01 DK053093), and D.R.B. (NIH R01 DK66370), 592, 2008 as well as grants from the PKD Foundation and the Harvard Center 18. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, Kido M, for PKD Research (NIH P50 DK074030). Hirokawa N: Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95: 829–837, 1998 19. McGrath J, Somlo S, Makova S, Tian X, Brueckner M: Two populations DISCLOSURES of node monocilia initiate left-right asymmetry in the mouse. Cell 114: None. 61–73, 2003 20. Wu G, D’Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, Maeda Y, Le TC, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S: Somatic in- activation of Pkd2 results in polycystic kidney disease. Cell 93: 177– REFERENCES 188, 1998 21. Pennekamp P, Karcher C, Fischer A, Schweickert A, Skryabin B, Horst J, 1. Wilson PD: Mouse models of polycystic kidney disease. Curr Top Dev Blum M, Dworniczak B: The ion channel polycystin-2 is required for left- – Biol 84: 311 350, 2008 right axis determination in mice. Curr Biol 12: 938–943, 2002 2. Parnell SC, Magenheimer BS, Maser RL, Rankin CA, Smine A, Okamoto 22. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, T, Calvet JP: The polycystic kidney disease-1 protein, polycystin-1, Brown EM, Quinn SJ, Ingber DE, Zhou J: Polycystins 1 and 2 mediate binds and activates heterotrimeric G-proteins in vitro. Biochem Biophys mechanosensation in the primary cilium of kidney cells. Nat Genet 33: Res Commun 251: 625–631, 1998 129–137, 2003 3. González-Perrett S, Kim K, Ibarra C, Damiano AE, Zotta E, Batelli M, 23. Field S, Riley KL, Grimes DT, Hilton H, Simon M, Powles-Glover N, Harris PC, Reisin IL, Arnaout MA, Cantiello HF: Polycystin-2, the protein Siggers P, Bogani D, Greenfield A, Norris DP: Pkd1l1 establishes left- mutated in autosomal dominant polycystic kidney disease (ADPKD), is a right asymmetry and physically interacts with Pkd2. Development 138: Ca2+-permeable nonselective cation channel. Proc Natl Acad Sci U S A 1131–1142, 2011 98: 1182–1187, 2001 24. Kamura K, Kobayashi D, Uehara Y, Koshida S, Iijima N, Kudo A, 4. Vassilev PM, Guo L, Chen XZ, Segal Y, Peng JB, Basora N, Babakhanlou Yokoyama T, Takeda H: Pkd1l1 complexes with Pkd2 on motile cilia and H, Cruger G, Kanazirska M, Ye Cp, Brown EM, Hediger MA, Zhou J: functions to establish the left-right axis. Development 138: 1121–1129, Polycystin-2 is a novel cation channel implicated in defective in- 2011 tracellular Ca(2+) homeostasis in polycystic kidney disease. Biochem 25. Lin F, Hiesberger T, Cordes K, Sinclair AM, Goldstein LS, Somlo S, Biophys Res Commun 282: 341–350, 2001 Igarashi P: Kidney-specific inactivation of the KIF3A subunit of kinesin-II 5. Delmas P, Nomura H, Li X, Lakkis M, Luo Y, Segal Y, Fernández- inhibits renal ciliogenesis and produces polycystic kidney disease. Proc Fernández JM, Harris P, Frischauf AM, Brown DA, Zhou J: Constitutive Natl Acad Sci U S A 100: 5286–5291, 2003 activation of G-proteins by polycystin-1 is antagonized by polycystin-2. 26. Yoder BK, Richards WG, Sweeney WE, Wilkinson JE, Avener ED, JBiolChem277: 11276–11283, 2002 Woychik RP: Insertional mutagenesis and molecular analysis of a new 6. Koulen P, Cai Y, Geng L, Maeda Y, Nishimura S, Witzgall R, Ehrlich BE, gene associated with polycystic kidney disease. Proc Assoc Am Somlo S: Polycystin-2 is an intracellular calcium release channel. Nat Physicians 107: 314–323, 1995 Cell Biol 4: 191–197, 2002 27. Takeda S, Yonekawa Y, Tanaka Y, Okada Y, Nonaka S, Hirokawa N: Left- 7. Zhou J: Polycystins and primary cilia: Primers for cell cycle progression. right asymmetry and kinesin superfamily protein KIF3A: New insights in Annu Rev Physiol 71: 83–113, 2009 determination of laterality and mesoderm induction by kif3A-/- mice 8. Hildebrandt F, Attanasio M, Otto E: Nephronophthisis: Disease analysis. JCellBiol145: 825–836, 1999 mechanisms of a ciliopathy. J Am Soc Nephrol 20: 23–35, 2009 28. Murcia NS, Richards WG, Yoder BK, Mucenski ML, Dunlap JR, Woychik 9. Simms RJ, Hynes AM, Eley L, Sayer JA: Nephronophthisis: A genetically RP: The Oak Ridge Polycystic Kidney (orpk) disease gene is required for diverse ciliopathy. Int J Nephrol 2011: 527137, 2011 left-right axis determination. Development 127: 2347–2355, 2000 10. Benzing T, Schermer B: Clinical spectrum and pathogenesis of neph- 29. Kitamura K, Miura H, Miyagawa-Tomita S, Yanazawa M, Katoh-Fukui Y, ronophthisis. Curr Opin Nephrol Hypertens 21: 272–278, 2012 Suzuki R, Ohuchi H, Suehiro A, Motegi Y, Nakahara Y, Kondo S, 11. Liu S, Lu W, Obara T, Kuida S, Lehoczky J, Dewar K, Drummond IA, Yokoyama M: Mouse Pitx2 deficiency leads to anomalies of the ventral Beier DR: A defect in a novel Nek-family kinase causes cystic kidney body wall, heart, extra- and periocular mesoderm and right pulmonary disease in the mouse and in zebrafish. Development 129: 5839–5846, isomerism. Development 126: 5749–5758, 1999 2002 30. Gage PJ, Suh H, Camper SA: Dosage requirement of Pitx2 for de- 12. Osmani SA, Pu RT, Morris NR: Mitotic induction and maintenance by velopment of multiple organs. Development 126: 4643–4651, 1999 overexpression of a G2-specific gene that encodes a potential protein 31. Lowe LA, Yamada S, Kuehn MR: Genetic dissection of nodal function in kinase. Cell 53: 237–244, 1988 patterning the mouse embryo. Development 128: 1831–1843, 2001 13. Nishimoto T, Eilen E, Basilico C: Premature of chromosome conden- 32. Kumar A, Lualdi M, Lewandoski M, Kuehn MR: Broad mesodermal and sation in a ts DNA- mutant of BHK cells. Cell 15: 475–483, 1978 endodermal deletion of Nodal at postgastrulation stages results solely 14. Bischoff FR, Ponstingl H: Catalysis of guanine nucleotide exchange on in left/right axial defects. Dev Dyn 237: 3591–3601, 2008 Ran by the mitotic regulator RCC1. Nature 354: 80–82, 1991 33. Yoder BK, Hou X, Guay-Woodford LM: The polycystic kidney disease 15. O’Connell MJ, Krien MJ, Hunter T: Never say never. The NIMA- proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized related protein kinases in mitotic control. Trends Cell Biol 13: 221–228, in renal cilia. JAmSocNephrol13: 2508–2516, 2002 2003 34. Mahjoub MR, Trapp ML, Quarmby LM: NIMA-related kinases defective 16. McCooke JK, Appels R, Barrero RA, Ding A, Ozimek-Kulik JE, Bellgard in murine models of polycystic kidney diseases localize to primary cilia MI, Morahan G, Phillips JK: A novel mutation causing nephronophthisis and centrosomes. JAmSocNephrol16: 3485–3489, 2005

110 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 100–112, 2013 www.jasn.org BASIC RESEARCH

35. Shiba D, Manning DK, Koga H, Beier DR, Yokoyama T: Inv acts as a 51. Fukui H, Shiba D, Asakawa K, Kawakami K, Yokoyama T: The ciliary molecular anchor for Nphp3 and Nek8 in the proximal segment of protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pro- primary cilia. Cytoskeleton (Hoboken) 67: 112–119, 2010 nephros morphogenesis and left-right establishment in zebrafish. FEBS 36. Smith LA, Bukanov NO, Husson H, Russo RJ, Barry TC, Taylor AL, Beier Lett 586: 2273–2279, 2012 DR, Ibraghimov-Beskrovnaya O: Development of polycystic kidney 52. Olbrich H, Fliegauf M, Hoefele J, Kispert A, Otto E, Volz A, Wolf MT, disease in juvenile cystic kidney mice: Insights into pathogenesis, ciliary Sasmaz G, Trauer U, Reinhardt R, Sudbrak R, Antignac C, Gretz N, abnormalities, and common features with human disease. JAmSoc Walz G, Schermer B, Benzing T, Hildebrandt F, Omran H: Mutations Nephrol 17: 2821–2831, 2006 in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto- 37. Sohara E, Luo Y, Zhang J, Manning DK, Beier DR, Zhou J: Nek8 regu- retinal degeneration and hepatic fibrosis. Nat Genet 34: 455–459, lates the expression and localization of polycystin-1 and polycystin-2. J 2003 Am Soc Nephrol 19: 469–476, 2008 53. Otto EA, Schermer B, Obara T, O’Toole JF, Hiller KS, Mueller AM, Ruf 38. George SH, Gertsenstein M, Vintersten K, Korets-Smith E, Murphy J, RG, Hoefele J, Beekmann F, Landau D, Foreman JW, Goodship JA, Stevens ME, Haigh JJ, Nagy A: Developmental and adult phenotyping Strachan T, Kispert A, Wolf MT, Gagnadoux MF, Nivet H, Antignac C, directly from mutant embryonic stem cells. Proc Natl Acad Sci U S A Walz G, Drummond IA, Benzing T, Hildebrandt F: Mutations in INVS 104: 4455–4460, 2007 encoding inversin cause nephronophthisis type 2, linking renal cystic 39. Wu G, Markowitz GS, Li L, D’Agati VD, Factor SM, Geng L, Tibara S, disease to the function of primary cilia and left-right axis determination. Tuchman J, Cai Y, Park JH, van Adelsberg J, Hou H Jr, Kucherlapati R, Nat Genet 34: 413–420, 2003 Edelmann W, Somlo S: Cardiac defects and renal failure in mice with 54. Kim I, Ding T, Fu Y, Li C, Cui L, Li A, Lian P, Liang D, Wang DW, Guo C, targeted mutations in Pkd2. Nat Genet 24: 75–78, 2000 Ma J, Zhao P, Coffey RJ, Zhan Q, Wu G: Conditional mutation of Pkd2 40. Tan SY, Rosenthal J, Zhao XQ, Francis RJ, Chatterjee B, Sabol SL, Linask causes cystogenesis and upregulates beta-catenin. J Am Soc Nephrol KL, Bracero L, Connelly PS, Daniels MP, Yu Q, Omran H, Leatherbury L, 20: 2556–2569, 2009 Lo CW: Heterotaxy and complex structural heart defects in a mutant 55. Yokoyama T, Copeland NG, Jenkins NA, Montgomery CA, Elder FF, mouse model of primary ciliary dyskinesia. JClinInvest117: 3742– Overbeek PA: Reversal of left-right asymmetry: A situs inversus muta- 3752, 2007 tion. Science 260: 679–682, 1993 41. Aune CN, Chatterjee B, Zhao XQ, Francis R, Bracero L, Yu Q, Rosenthal 56. Morishima M, Yasui H, Nakazawa M, Ando M, Ishibashi M, Takao A: J, Leatherbury L, Lo CW: Mouse model of heterotaxy with single ven- Situs variation and cardiovascular anomalies in the transgenic mouse tricle spectrum of cardiac anomalies. Pediatr Res 63: 9–14, 2008 insertional mutation, inv. Teratology 57: 302–309, 1998 42. Morgan D, Turnpenny L, Goodship J, Dai W, Majumder K, Matthews L, 57. Mochizuki T, Saijoh Y, Tsuchiya K, Shirayoshi Y, Takai S, Taya C, Gardner A, Schuster G, Vien L, Harrison W, Elder FF, Penman-Splitt M, Yonekawa H, Yamada K, Nihei H, Nakatsuji N, Overbeek PA, Hamada Overbeek P, Strachan T: Inversin, a novel gene in the vertebrate left- H, Yokoyama T: Cloning of inv, a gene that controls left/right asym- right axis pathway, is partially deleted in the inv mouse. Nat Genet 20: metry and kidney development. Nature 395: 177–181, 1998 149–156, 1998 58. Okada Y, Nonaka S, Tanaka Y, Saijoh Y, Hamada H, Hirokawa N: Ab- 43. Collignon J, Varlet I, Robertson EJ: Relationship between asymmetric normal nodal flow precedes situs inversus in iv and inv mice. Mol Cell 4: nodal expression and the direction of embryonic turning. Nature 381: 459–468, 1999 155–158, 1996 59. Basu B, Brueckner M: Cilia multifunctional organelles at the center of 44. Lowe LA, Supp DM, Sampath K, Yokoyama T, Wright CV, Potter SS, vertebrate left-right asymmetry. Curr Top Dev Biol 85: 151–174, 2008 Overbeek P, Kuehn MR: Conserved left-right asymmetry of nodal ex- 60. McQuinn TC, Miga DE, Mjaatvedt CH, Phelps AL, Wessels A: Cardio- pression and alterations in murine situs inversus. Nature 381: 158–161, pulmonary malformations in the inv/inv mouse. Anat Rec 263: 62–71, 1996 2001 45. Yoshioka H, Meno C, Koshiba K, Sugihara M, Itoh H, Ishimaru Y, Inoue 61. Slough J, Cooney L, Brueckner M: Monocilia in the embryonic mouse T, Ohuchi H, Semina EV, Murray JC, Hamada H, Noji S: Pitx2, a bicoid- heart suggest a direct role for cilia in cardiac morphogenesis. Dev Dyn type homeobox gene, is involved in a lefty-signaling pathway in de- 237: 2304–2314, 2008 termination of left-right asymmetry. Cell 94: 299–305, 1998 62. Bergmann C, Fliegauf M, Brüchle NO, Frank V, Olbrich H, Kirschner J, 46. Magenheimer BS, St John PL, Isom KS, Abrahamson DR, De Lisle RC, Schermer B, Schmedding I, Kispert A, Kränzlin B, Nürnberg G, Becker Wallace DP, Maser RL, Grantham JJ, Calvet JP: Early embryonic renal C, Grimm T, Girschick G, Lynch SA, Kelehan P, Senderek J, Neuhaus TJ, tubules of wild-type and polycystic kidney disease kidneys respond to Stallmach T, Zentgraf H, Nürnberg P, Gretz N, Lo C, Lienkamp S, cAMP stimulation with cystic fibrosis transmembrane conductance Schäfer T, Walz G, Benzing T, Zerres K, Omran H: Loss of nephrocystin- regulator/Na(+),K(+),2Cl(-) co-transporter-dependent cystic dilation. J 3 function can cause embryonic lethality, Meckel-Gruber-like syn- Am Soc Nephrol 17: 3424–3437, 2006 drome, situs inversus, and renal-hepatic-pancreatic dysplasia. Am J 47. Natoli TA, Gareski TC, Dackowski WR, Smith L, Bukanov NO, Russo RJ, Hum Genet 82: 959–970, 2008 Husson H, Matthews D, Piepenhagen P, Ibraghimov-Beskrovnaya O: 63. Trapp ML, Galtseva A, Manning DK, Beier DR, Rosenblum ND, Pkd1 and Nek8 mutations affect cell-cell adhesion and cilia in cysts Quarmby LM: Defects in ciliary localization of Nek8 is associated with formed in kidney organ cultures. Am J Physiol Renal Physiol 294: F73– cystogenesis. Pediatr Nephrol 23: 377–387, 2008 F83, 2008 64. Zalli D, Bayliss R, Fry AM: The Nek8 protein kinase, mutated in the 48. Pologruto TA, Yasuda R, Svoboda K: Monitoring neural activity and human cystic kidney disease nephronophthisis, is both activated and [Ca2+] with genetically encoded Ca2+ indicators. JNeurosci24: 9572– degraded during ciliogenesis. Hum Mol Genet 21: 1155–1171, 2012 9579, 2004 65. Park EY, Sung YH, Yang MH, Noh JY, Park SY, Lee TY, Yook YJ, Yoo KH, 49. Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu Roh KJ, Kim I, Hwang YH, Oh GT, Seong JK, Ahn C, Lee HW, Park JH: L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Cyst formation in kidney via B-Raf signaling in the PKD2 transgenic Svoboda K, Looger LL: Imaging neural activity in worms, flies and mice mice. JBiolChem284: 7214–7222, 2009 with improved GCaMP calcium indicators. Nat Methods 6: 875–881, 66. Liu P, Jenkins NA, Copeland NG: A highly efficient recombineering- 2009 based method for generating conditional knockout mutations. Ge- 50. Besschetnova TY, Kolpakova-Hart E, Guan Y, Zhou J, Olsen BR, Shah nome Res 13: 476–484, 2003 JV: Identification of signaling pathways regulating primary cilium 67. Joyner AL: Gene Targeting: A Practical Approach. Oxford, UK, Oxford length and flow-mediated adaptation. Curr Biol 20: 182–187, 2010 University Press, 2000

J Am Soc Nephrol 24: 100–112, 2013 Nek8 Disruption Causes Laterality and Renal Defects 111 BASIC RESEARCH www.jasn.org

68. Hahn WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM, SCFbeta-Trcp E3 complex. Mol Cell Biol 27: 6383–6395, DeCaprio JA, Weinberg RA: Enumeration of the simian virus 40 early 2007 region elements necessary for human cell transformation. Mol Cell Biol 72. Wilkinson DG: In Situ Hybridization: A Practical Approach, Oxford, UK, 22: 2111–2123, 2002 IRL Press, 1992 69. Besschetnova TY, Roy B, Shah JV: Imaging intraflagellar transport 73. Yu X, St Amand TR, Wang S, Li G, Zhang Y, Hu YP, Nguyen L, Qiu MS, Chen in mammalian primary cilia. Methods Cell Biol 93: 331–346, 2009 YP: Differential expression and functional analysis of Pitx2 isoforms in reg- 70. Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB: ulation of heart looping in the chick. Development 128: 1005–1013, 2001 Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr Biol 12: R378–R380, 2002 71. Tian Y, Kolb R, Hong JH, Carroll J, Li D, You J, Bronson R, Yaffe MB, This article contains supplemental material online at http://jasn.asnjournals. Zhou J, Benjamin T: TAZ promotes PC2 degradation through a org/lookup/suppl/doi:10.1681/ASN.2012050490/-/DCSupplemental.

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