Disruption of the mouse Jhy gene causes abnormal ciliary microtubulepatterning and juvenile hydrocephalus

Oliver K. Appelbe a, Bryan Bollman a, Ali Attarwala a, Lindy A. Triebes a,Hilmarie Muniz-Talavera a, Daniel J. Curry b,1, Jennifer V. Schmidt a,nQ1

a Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, United Statesb Department of Neurosurgery, University of Chicago, Chicago, IL, United States

a r t i c l e i n f o

Article history:Received 21 December 2012Received in revised form10 June 2013Accepted 5 July 2013

Keywords:MouseHydrocephalusCilia4931429I11RikJhy

a b s t r a c t

Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of thebrain, affects one of every 1000 children born today, making it one of the most common humandevelopmental disorders. Genetic causes of hydrocephalus are poorly understood in humans, but animalmodels suggest a broad genetic program underlying the regulation of CSF balance. In this study, therandom integration of a transgene into the mouse genome led to the development of an early onset andrapidly progressive hydrocephalus. Juvenile hydrocephalus transgenic mice (JhylacZ) inherit communicat-ing hydrocephalus in an autosomal recessive fashion with dilation of the lateral ventricles observed asearly as postnatal day 1.5. Ventricular dilation increases in severity over time, becoming fatal at 4–8weeks of age. The ependymal cilia lining the lateral ventricles are morphologically abnormal and reducedin number in JhylacZ/lacZ brains, and ultrastructural analysis revealed disorganization of the expected 9+2microtubule pattern. Rather, the majority of JhylacZ/lacZ cilia develop axonemes with 9+0 or 8+2 microtubulestructures. Disruption of an unstudied gene, 4931429I11Rik (now named Jhy) appears to underlie thehydrocephalus of JhylacZ/lacZ mice, and the Jhy transcript and protein are decreased in JhylacZ/lacZ mice.Partial phenotypic rescue was achieved in JhylacZ/lacZ mice by the introduction of a bacterial artificialchromosome (BAC) carrying 60–70% of the JHY protein coding sequence. Jhy is evolutionarily conservedfrom humans to basal vertebrates, but the predicted JHY protein lacks identifiable functional domains.Ongoing studies are directed at uncovering the physiological function of JHY and its role in CSFhomeostasis.

& 2013 Elsevier Inc. All rights reserved.

Introduction

The cerebral spinal fluid (CSF) within the ventricles of the brainfunctions to cushion, nourish and cleanse the brain tissues, andacts as a carrier for key growth factors and signaling molecules(Davson and Segal, 1996; Rodriguez, 1976; Wood, 1983). CSF iscontinually produced by the choroid plexus tissue within theventricles, and flows from the lateral and third ventricles throughthe narrow aqueduct of Sylvius into the fourth ventricle (Welch,1963; Worthington and Cathcart, 1966; Yamadori and Nara, 1979).CSF flow is accomplished by the movement of cilia on the surfaceof the ependymal cells lining the ventricles, and likely also bypulsatile changes in intracranial blood flow (Bhadelia et al., 1997;Greitz, 1993; Quencer et al., 1990). From the fourth ventricle, thefluid exits the brain, moving into the spinal canal and the

subarachnoid space. CSF production is balanced by its removal;once thought to occur only through the arachnoid villi into thebloodstream, it is now believed that both the lymphatic systemand the ventricular ependymal cells play a role in CSF uptake (Kohet al., 2006; Oi and Di Rocco, 2006; Weller et al., 1992).

An increase in the amount of CSF within the closed space of thebrain results in the disease known as hydrocephalus. Congenitalhydrocephalus is one of the most common human developmentaldisorders, and may present nonsyndromically or in conjunctionwith a number of developmental malformations. Population-basedstudies estimate an incidence of 0.4 to 0.8 in 1000 children bornwith hydrocephalus in the absence of any neural tube defect(Blackburn and Fineman, 1994; Christensen et al., 2003;Schrander-Stumpel and Fryns, 1998; Stoll et al., 1992). Physiologi-cally, hydrocephalus may be caused by (1) overproduction of CSF,(2) failure to resorb CSF, or (3) a blockage that prevents flowthrough the ventricles. Hydrocephalus is categorized as eithercommunicating, in which there is no physical blockage to CSF flow,or noncommunicating, in which CSF flow is blocked, usually at theaqueduct. As the volume of CSF in the ventricles increases, the

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Developmental Biology

0012-1606/$ - see front matter & 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ydbio.2013.07.003

n Corresponding author. Fax: +1 312 413 2691.E-mail address: jvs@uic.edu (J.V. Schmidt).1 Current address: Department of Neurosurgery, Texas Children!s Hospital,

Houston, TX, United States.

Please cite this article as: Appelbe, O.K., et al., Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning andjuvenile hydrocephalus. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.003i

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

pressure on surrounding brain tissues leads to neuronal cell death.Left untreated, progressive tissue destruction leads to physical andcognitive decline and eventual death (Gadsdon et al., 1979).

Congenital hydrocephalus, in which the disease presents at orshortly after birth, is often associated with subarachnoid hemor-rhage, where residual blood products are believed to occlude thearachnoid (Rudas et al., 1998). It has been estimated that 60% of thecases of congenital hydrocephalus in human infants are acquired,with an identifiable pathology, while the other 40% have no knowncause and are likely genetic in origin (Haverkamp et al., 1999). Oneknown genetic cause of congenital hydrocephalus in humans ismutation of the gene encoding the L1CAM cell adhesion protein,although studies show a strong heritable component to the devel-opment of hydrocephalus (Castro-Gago et al., 1996; Chalmers et al.,1999; Chow et al., 1990; Chudley et al., 1997; Hamada et al., 1999;Koh and Boles, 1998; Munch et al., 2012; Portenoy et al., 1984;Rosenthal et al., 1992; Verhagen et al., 1998; Vincent et al., 1994).

The study of animal models has uncovered numerous mechanismsthat may underlie the development of hydrocephalus. For example,abnormal differentiation of the arachnoid layer in mice deficient in theforkhead gene Foxc1 leads to communicating hydrocephalus due to adecrease in CSF absorption (Green, 1970; Kume et al., 1998). Deletionof a genomic segment encompassing the human FOXC1 gene has beenassociated with hydrocephalus in four human patients (Kume et al.,1998). Abnormal development of the subcommissural organ, over-production of CSF by the choroid plexus, ependymal denudation ordisorganization, and defects in ependymal cilia have also been shownto cause hydrocephalus in mouse and rat models (Banizs et al., 2005;Batiz et al., 2006; Blackshear et al., 2003; Davy and Robinson, 2003;Feng et al., 2009; Ibanez-Tallon et al., 2004; Jones and Bucknall, 1988;Krebs et al., 2004; Lechtreck et al., 2008; Lindeman et al., 1998; Sapiroet al., 2002; Tullio et al., 2001; Wilson et al., 2010).

Enhancer trapping, the random integration of a minimal promoter-reporter transgene, can localize genes active in specific tissues or atspecific times during development (Allen et al., 1988). Insertionalmutagenesis during the generation of transgenic mice is common, andenhancer traps may confer such mutations when they not only “trap”,but also inactivate, a gene or its regulatory elements. A promotermapping transgene generated in our laboratory inadvertently func-tioned as an enhancer trap, conferring widely varying lacZ expressionpatterns across independent transgenic lines. One of these linesshowed lacZ expression in the embryonic and adult brain, and ashomozygotes, the mice developed a rapidly progressing juvenilehydrocephalus. These Juvenile hydrocephalus mice (formallyJhyTg(Dlk1-lacZ)1Jvs, here abbreviated JhylacZ) show overt doming of theskull by 1 week after birth, and die or must be sacrificed by 4–8 weeksof age. Histological analysis shows the open aqueduct indicative ofcommunicating hydrocephalus, and electron microscopy identifiedaltered microtubule organization of the ependymal cilia. Mapping ofthe transgene integration site found that it disrupted the previouslyunstudied gene 4931429I11Rik (now named Jhy). The integration andan associated deletion remove a splice acceptor site within the Jhygene, suggesting reduced function of Jhy is causative for the pheno-type. The Jhy gene is conserved across vertebrates, but has nohomology to other genes in the mouse genome, and carries noidentifiable functional domains. This manuscript provides a detaileddescription of the JhylacZ/lacZ mouse phenotype; characterization of theJhy gene is ongoing.

Methods

JhylacZ mice

The Juvenile hydrocephalus transgene (JhylacZ) was generatedfrom the promoterless lacZ vector pNASS-β (Clontech, Mountain

View, CA). A genomic fragment from !248 bp to +50 bp relative tothe Dlk1 transcriptional initiation site (Osoegawa et al., 2000) wasisolated and cloned into the vector. The transgene (!248Dlk1lacZ),including the Dlk1 promoter, SV40 splice donor-acceptor site, lacZgene, and polyadenylation signal, was then excised, gel purified,and injected into FVB/N fertilized mouse eggs with the assistanceof the University of Illinois at Chicago Transgenic ProductionService. Offspring were genotyped by PCR using primers spanningthe junction between the Dlk1 promoter (OL410, 5!-GGGGACGA-CAGTACGAAAAGGC-3!) and the lacZ gene (OL411, 5!-GTATCGGCCT-CAGGAAGATCGC-3!). DNA for genotyping was obtained from tailclips, digested by proteinase K overnight, phenol:chloroformextracted, and ethanol precipitated. Twelve transgenic lines wereestablished on an FVB/N background and analyzed for lacZexpression. The JhylacZ line is maintained by intercrossing JhylacZ/+

animals. JhylacZ mice were genotyped using two sets of primersthat amplified either the wild type allele (OL1408, 5!-AAGCTCTGTCTGGGCTCACAATCT-3!; OL1409, 5!-TGACCTCTTGGGACATTGC-CTGAT-3!) or the JhylacZ allele (OL1420, 5!-TCCAGTTCAACATCAGCCGC-TACA-3!; OL1395, 5!-TTAGAAGCGTTCGTTTGTGCAGCC-3!). PCR wasperformed in an Eppendorf Mastercycler (Hauppauge, NY) as follows:95 1C for 5 min; 35 cycles of 94 1C for 30 s, 62 1C for 30 s, and 72 1Cfor 1 min; and a final step of 72 1C for 10 min.

!-Galactosidase assay for lacZ expression

Brains were dissected at postnatal day 0.5 (P0.5) and P5, testeswere dissected from adult mice, and both were immediately fixedin 4% paraformaldehyde in 1X PBS for 1 h on ice. Followingfixation, tissues were washed in wash buffer (2 mM MgCl2, 0.01%deoxycholic acid, 0.02% NP-40, in PBS) three times for 30 min eachat room temperature (RT). Expression of lacZ was detected asβ-galactosidase staining using the detection solution (1 mg/ml X-gal in DMF, 5 mM potassium ferricyanide, 5 mM potassium ferro-cyanide, 2 mM MgCl2, 0.01% deoxycholic acid, 0.02% NP-40, inPBS), incubating overnight in the dark at RT. The next day, tissueswere washed three times in 1" PBS, dehydrated in solutions ofincreasing ethanol concentration, and infused with paraffin waxusing a Shandon Citadel 1000 tissue processor (Fisher Scientific,Pittsburgh, PA, USA). Processed tissues were embedded in blocksof paraffin using a Shandon Histocentre 2 embedding machine(Fisher Scientific, Pittsburgh, PA). Embedded tissues were cut into10 μm sections using a microtome (Leica Microsystems RM2125,Bannockburn, IL, USA) and background stained with 10% eosin for1 min. Coverslips were applied using Permount (Fisher Scientific,Pittsburgh, PA) and images were taken on a Leica MZFLIII dissect-ing microscope (Wetzlar, Germany).

Magnetic resonance imaging

MRI was performed at the University of Chicago MagneticResonance Imaging & Spectroscopy Research Laboratory. Jhy+/+

and JhylacZ/lacZ mice at P6 and P13 were sacrificed by CO2 inhalationapproximately one hour prior to imaging. Imaging was performedusing a Bruker scanner equipped with a 4.7 T magnet and acustom-made birdcage coil for juvenile mice. Coronal T2-weighted axial spin echo images were collected using the para-meters: repetition time 3000 ms, echo time 60 ms, array size256"256, field of view 2.0 cm.

Genomic localization and sequence analysis

The BD GenomeWalker Universal Kit (Clontech, Mountain View,CA) was used to map the integration site of the JhylacZ transgene.The GenomeWalker protocol was followed with the followingspecifications. A library created from DraI digestion of genomic

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Please cite this article as: Appelbe, O.K., et al., Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning andjuvenile hydrocephalus. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.003i

tail DNA was used for mapping the distal transgene integrationboundary. Following the purification of digested DNA and ligation ofadaptors, nested PCR was performed with the following primers:outer (OL834, 5!-CCCGTTTTTCCCGATTTGGCTACATGACA-3!;AP1, 5!-GTAATACGACTCACTATAGGGC-3!) and inner (OL836, 5!-CAACCGCTGTTTGGTCTGCTTTCTGACAA-3!; AP2, 5!-ACTATAGGG-CACGCGTGGT-3!). The proximal boundary of the transgene integra-tion was mapped using the following primers: OL1368, 5!-AACACTTTCATGGATCCTGAGGACA-3!; OL1618, 5!-GTAACCGTG-CATCTGCCAGTTTG-3!. PCR products were sequenced by the Uni-versity of Illinois at Chicago Research Resources Center DNAServices facility (Chicago, IL). Cross-species Jhy cDNA and proteinalignments were performed in Geneious v5.6.6 (Biomatters Ltd)using Geneious and ClustalW alignments, respectively, with thedefault parameters.

Quantitative RT-PCR

Total RNA was extracted from P0.5 mouse brains using LiCl-urea precipitation (Auffray and Rougeon, 1980), and DNA contam-ination was removed using TURBO DNA-free (Ambion, Austin, TX).Reverse transcription was performed with 1 μg of total RNA usingSuperscript III reverse transcriptase (Invitrogen, Carlsbad, CA). ThecDNA was diluted 1:10, and 2 μl was used for PCR analysis. Controlreactions were performed without reverse transcriptase. Quanti-tative RT-PCR (qRT-PCR) was performed using the DNA EngineOpticon 2 (MJ Research, Hercules, CA) with the following primers:Jhy, OL1366, 5!-AGCCAACAACACTAACAGGGAAGA-3! and OL1524,5!-TCCAGTTGGGATCATATCGGAGGT-3! that span exons 2–3; Bsx,OL1325, 5!-CAGAACCGGCGGATGAAGCATAAA-3! and OL1326, 5!-ACTTCGTCCTCGGGCTCAGTAA that lie within exon 3; Crtam,OL1114, 5!-AGCACACGTGGTATGGAAGAAGGA and OL1115, 5!-TTTGGCTCCCGAGTATAACTGCGT that span exons 8–10; !-actin, OL1112,5!-TCTTGGGTATGGAATCCTGTGGCA-3! and OL1113, 5!-TCTCCTTC-TGCATCCTGTCAGCAA-3!. For all primer pairs qRT-PCR was per-formed using SYBR GreenER (Invitrogen, Carlsbad, CA) under thefollowing conditions: 50 1C for 2 min; 95 1C for 10 min; followedby 40 cycles of 94 1C for 15 s, 62 1C for 30 s, 72 1C for 15 s. Amelting curve ranging from 60 1C to 95 1C was run after eachreaction to test for primer dimerization or the formation ofmultiple products.

TgBAC rescue mice

The BAC clone RP23-273J15 was obtained from the Children!sHospital Oakland Research Institute (Oakland, CA). BAC DNAwas linearized with BspEI and prepared for injection by phenol:chloroform extraction, ethanol precipitation, and resuspension inmicroinjection buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA,30 μM spermine, 70 μM spermidine, 100 mM NaCl). The linearizedBAC was microinjected into FVB/N fertilized mouse eggs with theassistance of the University of Illinois at Chicago TransgenicProduction Service and 38 offspring were produced. Pups weregenotyped for the BAC from tail DNA using the primer set: OL1183,5!-CGACTCAAGCCTTCGCGAAAGAAA-3!; OL1561, 5!-CGTGTTTGA-AGTGATCAGCGGCTT-3!. Five of the 38 transgenic mice werepositive for the BAC, and one expressing transgenic line wasgenerated on the FVB/N background. Formally Tg(RP23-273J15)1Jvs, this line is here abbreviated TgBAC.

Scanning electron microscopy

Jhy+/+, JhylacZ/+, and JhylacZ/lacZ littermate P5 brains were dis-sected and fixed in Karnovsky!s fixative (2.5% glutaraldehyde, 2.0%paraformaldehyde, 0.1 M sodium cacodylate buffer pH 7.4) for4 days at RT. Dissecting the left and right hemispheres away from

the midbrain and removing the hippocampus exposed the lateralventricles. Brains were washed three times in PBS at RT, thendehydrated in a graded ethanol series on ice with the 100% ethanolsolution containing molecular sieves. The preparations were driedin a Bal-Tec CPD 030 Critical Point Dryer (Bal-Tec AG, Fürstentum,Liechtenstein), coated with gold/palladium using a DentonVacuum Desk IV Sputter Coater (Denton Vacuum LLC, Moores-town, NJ) set at 48% power, and imaged directly on a JEOL 5600 LVscanning electron microscope (JEOL, Peabody, MA). The accelerat-ing voltage was set at 15 kV, with the higher magnification imagestaken at 20 kV.

Transmission electron microscopy

Jhy+/+ and JhylacZ/lacZ brains were dissected at P5 and immersed inKarnovsky!s fixative for 7 days. The lateral ventricles were isolatedand further fixed in Karnovsky!s for an additional 24 h. The tissue waswashed in three changes of 0.1 M sodium cacodylate buffer pH7.4 and immersed in 1% osmium tetroxide in 0.1 M sodium cacodylatebuffer pH 7.4 for two hours at RT. The tissue was again washed in0.1 M sodium cacodylate buffer pH 7.4 and then dehydrated usingincreasing concentrations of ethanol. The tissue was infiltrated over-night with 1 part 100% ethanol and 1 part Spurr!s resin, and twoadditional changes with pure Spurr!s resin were made over a 24-hperiod. Finally, the tissue was embedded with Spurr!s resin inEppendorf tubes, and polymerized at 70 1C for 48 h. The polymerizedblocks were trimmed and faced off at 1 μm sections using a Reichert-Jung Ultracut E Ultramicrotome (Leica Microsystems, Buffalo Grove,IL). The 1 μm sections were placed on glass slides, dyed with 1%toluidine blue, and viewed to determine the region to be used forimaging. Next, 80 nm sections were cut and picked up onto 200 hexmesh nickel parlodion carbon coated grids and allowed to completelydry. The sections were stained for 5 min in lead citrate, followed bythree washes in deionized water. Once dried, the sections werestained for 45 min with uranyl acetate (4% aqueous), followed bythree washes in deionized water. The sections were dried, and stainedfor an additional 5 min with lead citrate, followed by three washes indeionized water, and allowed to completely dry. The sections wereviewed using a JEOL 1200EX transmission electron microscope (JEOL,Peabody, MA, USA), and areas of interest were photographed.

Statistical analysis

Two-tailed Student!s t-tests were used to calculate the signifi-cance of data obtained in qRT-PCR experiments. Standard error ofthe mean was used to calculate error bars.

Immunofluorescence

P5 mouse brains were fixed in Bouin!s fixative for 18–24 h atRT, then cleared and stored in 70% ethanol. Tissues were thendehydrated, embedded in paraffin and sectioned with a micro-tome to 8 μm. Standard immunofluorescence techniques wereused, with variations as follows. For JHY detection, sections weredewaxed with xylene and rehydrated through a series of ethanolwashes. Heat induced antigen retrieval (0.3% Sodium Citrate, 0.05%Tween-20 pH 6.0) was performed followed by washing in PBS.Slides were blocked using 5% donkey normal serum, 1% BSA, 0.5%Tween-20, 0.75% glycine in PBS for 1 h at RT, and incubated at 4 1Covernight with primary antibody against JHY (1:10; Sigma-Aldrich,HPA039612). The sections were then incubated with Alexa Fluor647-conjugated secondary antibody for 2 h at RT (1:250; JacksonImmunoResearch Laboratories, West Grove, PA). Finally, the slideswere DAPI stained (D1306; Life Technologies, Grand Island, NY.)and mounted using Vectashield mounting medium (H1000; VectorLaboratories, Burlingame, CA).

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Please cite this article as: Appelbe, O.K., et al., Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning andjuvenile hydrocephalus. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.003i

Results

Enhancer trapping identifies a gene required for CSF balance

An experiment was designed to identify regulatory elements ofthe mouse Delta-like 1 (Dlk1) gene (Fay et al., 1988; Laborda et al.,1993; Okamoto et al., 1997; Smas and Sul, 1993). Varying lengths ofthe Dlk1 upstream region were linked to a lacZ reporter gene andinjected into fertilized FVB/N mouse eggs, generating 12 indepen-dent transgenic lines. When analyzed for lacZ expression duringmidgestation, embryos from the different lines showed widelyvarying patterns of expression, none of them resembling the wellcharacterized pattern of Dlk1 (data not shown). Enhancer trappingcan occur unintentionally, when a transgene that is believed tocarry an enhancer either has no enhancer, or it is overwhelmed bystronger regulatory elements at the site of integration. Analysis ofthe Dlk1 upstream region subsequently found that no develop-mental enhancers lie within the transgene sequences; the Dlk1-lacZ transgenes functioned instead as enhancer traps (Rogers et al.,2012; Yevtodiyenko et al., 2004).

One transgenic line, examined as whole mount embryos atembryonic day 11.5 (e11.5), showed lacZ expression in a small

region on the dorsal aspect of the head (Fig. 1A–C). Histologicanalysis identified the expressing structure as the epiphysis, theroof of the diencephalon that will form the pineal gland (Fig. 1C).Intercrossing the transgenic animals showed that mice homozy-gous for the transgene develop progressive and ultimately fataljuvenile hydrocephalus (Fig. 1D and E). None of the other trans-genic lines exhibited any degree of hydrocephalus, suggesting thephenotype results from an insertional mutation generated by thetransgene, rather than expression of the transgene itself. Thetransgene integration therefore trapped, and likely altered thefunction of, a gene required for proper CSF balance in thedeveloping mouse brain. The transgenic mouse line has thereforebeen named JhylacZ for Juvenile hydrocephalus.

JhylacZ/lacZ mice show early and progressive hydrocephalus

Heterozygous JhylacZ/+ mice appear normal in all aspects, andshowed no evidence of hydrocephalus. Intercrossing JhylacZ/+

animals gave offspring that appeared normal at birth, and allgenotypes were found in the expected ratios. As the JhylacZ/lacZ

homozygous mice grew, however, 63% began to display the domedhead indicative of hydrocephalus between 2 and 5 weeks after

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Fig. 1. JhylacZ/lacZ mice develop hydrocephalus. (A) JhylacZ/lacZ embryo at e11.5 showing lacZ expression in the epiphysis of the diencephalon. (B) Enlarged view of lacZexpression in A. (C) Saggital section of diencephalon lacZ expression in e11.5 JhylacZ/lacZ embryo. (D) Jhy+/+ (left) and JhylacZ/lacZ littermates (right) at 3 weeks of age, showingdoming of the skull and ataxia in the JhylacZ/lacZ mouse. (E) Hemorrhaging beneath the skull is visible in a 3 week old JhylacZ/lacZ mouse (top) compared to a Jhy+/+ littermate(bottom). (F) and (G) Hematoxylin/eosin stained sections of Jhy+/+ (F) and JhylacZ/lacZ (G) brains at P5, showing ventricular dilation.

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Please cite this article as: Appelbe, O.K., et al., Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning andjuvenile hydrocephalus. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.003i

birth (Figs. 1D, E and 2A). The enlargement of the head progressesrapidly, and as the disease worsens the mice become ataxic andbegin to lose weight, likely from motor deficits that interfere withfeeding (Fig. 1D). Intracranial hemorrhaging can often be observedexternally in the albino FVB/N mice, though this occurs relativelylate in the course of the disease and appears to be secondary to thehydrocephalus (Fig. 1E). Histological analysis of the brains ofJhylacZ/lacZ animals at P5 shows dramatically enlarged lateralventricles and loss of neuronal tissue (Fig. 1F and G). All of theseearly onset hydrocephalic mice have died, or been sacrificed forhumane reasons, by 4–8 weeks of age (Fig. 2B). A small percentageof JhylacZ/lacZ mice do not show the skull doming of early hydro-cephalus, but begin to display the related ataxia and weight loss at5–6 weeks of age (Fig. 2C). The lack of a skull phenotype isexpected in these late onset animals, as the fusion of the anteriorfrontal cranial suture between 3 and 6 weeks of age prevents skullenlargement beyond this point (Bradley et al., 1996). These lateonset mice were followed until 12 weeks of age and all survived tothis time point, suggesting a milder course of the disease. Overall,74% of JhylacZ/lacZ mice develop hydrocephalus, the majority asjuveniles.

JhylacZ/lacZ mice show normal brain patterning

Juvenile hydrocephalus can be secondary to abnormal braindevelopment or patterning, when malformations may physicallyobstruct CSF flow (Partington, 2001; Pattisapu, 2001). Develop-mental malformation syndromes are characterized by grossdefects in brain structure, or by more subtle disorganization ofthe cortical layers. To more closely analyze the neuronal develop-ment of JhylacZ/lacZ mice, brains were examined by histologicalanalysis at e18.5, P0.5 and P5. JhylacZ/lacZ brains appear structurallynormal at e18.5 with normal ventricular size and shape and opencommunication between the ventricles (data not shown). Serialsectioning was then performed through entire Jhy+/+ and JhylacZ/lacZ

mouse brains at P0.5, prior to the onset of hydrocephalus (Fig. 3A–H).No changes were seen in comparison to Jhy+/+ littermates, and allexisting brain structures appeared correctly patterned. By P1.5mild hydrocephalus is evident in many JhylacZ/lacZ brains (data notshown), and at P5 the lateral and third ventricles of JhylacZ/lacZ miceare significantly larger than their Jhy+/+ littermates (Fig. 1F and G).The aqueduct and fourth ventricle do not appear enlarged at anystage observed, and the aqueduct remains patent at P0.5, asvisualized in transverse and coronal sections (Fig. 3I and datanot shown). These data indicate that JhylacZ mice suffer fromcommunicating hydrocephalus, in the absence of any develop-mental malformation.

To examine the progression of the disease in the intact animal,magnetic resonance imaging (MRI) was used to view the brains ofJhy+/+ and JhylacZ/lacZ littermates (Fig. 3J–M). At P6, dilation of thelateral ventricles can be seen in JhylacZ/lacZ brains as compared toage matched Jhy+/+ brains (Fig. 3J and K). At P13, the ventriculardilation in JhylacZ brains has increased, with a corresponding loss ofbrain tissue and thinning of the overlying cortex (Fig. 3L and M).The dorsal region of the third ventricle is noticeably dilated at P13(data not shown); the ventral region of the third ventricle and thefourth ventricle were not visualized in this analysis.

JhylacZ/lacZ hydrocephalus is nonsyndromic

Hydrocephalus, in both mouse models and human patients,may occur as one component of certain generalized ciliopathies(Baker and Beales, 2009; Lee, 2011). Individuals suffering fromthese ciliary dysfunction syndromes display other cilia-relatedphenotypes that include situs inversus, infertility and recurrentrespiratory infections (Ferkol and Leigh, 2012). No JhylacZ/lacZ mice

were found to display situs inversus, and they showed no evidenceof respiratory infections during several years of maintenance.Histological analysis was performed on all major organ systemsof Jhy+/+ and early onset JhylacZ/lacZ mice at 4 weeks of age (Fig. 3N–Qand data not shown). Particular attention was paid to those tissuesinvolved in ciliary-related disease syndromes, such as the lungs,kidney and testis. With the exception of the testes, no alterationswere found in any of the tissues examined. Overall testis structurewas normal in JhylacZ/lacZ mice, but there appeared to be reducednumbers of mature, flagellated sperm in the lumen of many

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Fig. 2. JhylacZ/lacZ hydrocephalus is rapid and progressive. (A) JhylacZ/lacZ mice(squares) develop outwardly visible hydrocephalus with a penetrance of 74%, withmost mice affected by 5 weeks of age. No JhylacZ/+ mice (diamonds) developedhydrocephalus or died during the 12 week monitoring period. (B) Most JhylacZ/lacZ micedie by 4–8 weeks of age. (C) Early onset hydrocephalus occurs in 63% of JhylacZ/lacZ

mice, with an additional 11% developing a late onset milder form of the disease.

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tubules (Fig. 3N and O). As one example of an unaffected tissue,the structure of the kidney was unaltered in JhylacZ/lacZ mice ascompared to Jhy+/+ (Fig. 3P and Q). The effects of the JhylacZ

mutation are therefore limited to the brain and the testes.Fertility can be difficult to analyze in early lethal mutations, and

most early onset JhylacZ/lacZ mice are quite debilitated by breeding age.No early onset JhylacZ/lacZ mice have produced pregnancies, but two ofthe late onset animals have given offspring. The longest survivinghydrocephalic JhylacZ/lacZ mouse, a male, fathered a litter of 4 pups withan FVB/N female at 2 months of age. One female JhylacZ/lacZ animal gavebirth to a litter of six pups at 3 months of age when crossed to a JhylacZ/+ male. The JhylacZ/lacZ offspring of this JhylacZ/lacZ female showed nophenotypic differences from JhylacZ/lacZ pups of a JhylacZ/+ female. JhylacZ/lacZ mice are therefore fertile, but males may show subfertility due to

reduced sperm numbers, and most animals of both sexes do not breeddue to the rapid progression of the hydrocephalus phenotype.

The JhylacZ reporter is expressed in the brain

The expression pattern of an enhancer trap transgene inte-grated into the mouse genome typically reflects that of a gene orgenes near the integration site. The integration of the JhylacZ

transgene within the Jhy gene suggests that lacZ may serve as aproxy for the expression of Jhy and/or other genes in the region.To further characterize the transgene expression pattern, lacZ expres-sion was analyzed at P0.5 and P5. At P0.5, lacZ expression is visible inthe pineal gland (Fig. 4A, B, E and F), in the hypothalamus (Fig. 4A, B,M and N), and in the ependymal cells of the aqueduct of Sylvius

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Fig. 3. JhylacZ/lacZ brains show normal development and patterning. (A)–(H) Coronal sections of Jhy+/+ and JhylacZ/lacZ brains at P0.5 show normal morphology and patterning.(I) Transverse section of JhylacZ/lacZ brain at P0.5 shows a patent aqueduct. The inset shows an enlarged image of the aqueduct. (J)–(M) Magnetic resonance imaging of Jhy+/+

and JhylacZ/lacZ brains. (J) and (K) MRI at P6 shows significant dilation of the lateral ventricles of the JhylacZ/lacZ mouse (K) as compared to a Jhy+/+ littermate (J). (L) and (M) MRIat P13 shows more pronounced ventricular dilation with loss of brain tissue. (N)–(Q) Adult testis ((N) and (O)) show reduced numbers of mature sperm, while kidney ((P) and(Q)) show normal development.

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(Fig. 4Q and R). At P5, expression is similarly observed in the pineal(Fig. 4C, D, G and H), the hypothalamus (Fig. 4C, D, O and P) and theependyma of the aqueduct of Sylvius (Fig. 4S and T). At this stageexpression is also seen in the choroid plexus of the third ventriclebut not the choroid plexus of the lateral or fourth ventricles (Fig. 4Kand L). Expression of the lacZ reporter within the developing brain isconsistent with a causative role for the transgene integration in thehydrocephalus of JhylacZ/lacZ mice. The choroid plexus and ependymalcells both play key roles in CSF balance, with the choroid responsiblefor producing the bulk of the CSF, while ependymal cilia regulate CSFflow and maintain a patent aqueduct (Bruni, 1998).

Ependymal cilia are morphologically abnormal in JhylacZ/lacZ mice

The motile cilia of the ependymal cells lining the ventricularsurfaces play a role in maintaining proper CSF flow through theventricular system. Defects in ciliary structure or function underlieseveral mouse models of hydrocephalus as well as certain humancases (al-Shroof et al., 2001; Batiz et al., 2006; Davy and Robinson,2003; Feng et al., 2009; Ibanez-Tallon et al., 2004; Sapiro et al., 2002;Tullio et al., 2001; Wessels et al., 2003). Scanning electron microscopy(SEM) was used to inspect the developing cilia of P5 JhylacZ/lacZ miceand their Jhy+/+ littermates. The P5 time point was chosen with the

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Fig. 4. JhylacZ transgene reporter expression in the brain. JhylacZ transgene expression was examined in Jhy+/+ and JhylacZ/lacZ mice at P0.5 and P5. (A)–(D) Expression of thelacZ reporter is observed in the pineal gland and hypothalamus at P0.5 and P5. Dilation of the lateral ventricles can be seen in JhylacZ/lacZ mice at P5. (E)–(H) Highermagnification of the pineal gland lacZ expression, and lack of expression in the subcommissural organ. (I)–(L) Expression of lacZ is seen in the choroid plexus of the thirdventricle at P5, but not in the choroid plexus or ependyma of the lateral ventricles. At P5 dilation of the third ventricle is seen in JhylacZ/lacZ mice. (M)–(P) Higher magnificationof the hypothalamus lacZ expression. (Q)–(T) Expression of lacZ in the ependyma lining the aqueduct of Sylvius. Structures are indicated as follows: A, aqueduct of Sylvius; C,choroid plexus; H, hypothalamus; L, lateral ventricle; P, pineal gland; S, subcommissural organ; 3, third ventricle.

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goal of observing any ciliary changes that may be causative for thehydrocephalus, while avoiding the effects of morphological damageand neuronal loss found at later stages. Opening the ventricles of Jhy+/+

animals invariably caused breakage at the attachment point that joinsthe ventricular walls (Fig. 5A, arrow). Early ventricular enlargement inJhylacZ/lacZ brains prior to attachment leads to a loss of this adhesion

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Fig. 5. JhylacZ/lacZ ependymal cilia exhibit structural abnormalities. (A)–(B) SEM was used to visualize the ultrastructure of the ependymal cilia in P5 Jhy+/+ (A) and JhylacZ/lacZ

(B) lateral ventricles. Caudal is to the left. (C)–(D) Higher magnification images of the ependymal cilia in P5 Jhy+/+ (C) and JhylacZ/lacZ (D) lateral ventricles. (E)–(F) Highmagnification images of the ependymal cilia in P5 Jhy+/+ (E) and JhylacZ/lacZ (F) lateral ventricles. (G)–(I) Representative TEM images of Jhy+/+ 9+2 (G), and JhylacZ/lacZ 9+0(H) and 8+2 (I) axonemes. (J) TEM image showing partial displacement of a peripheral ring microtubule doublet towards the center of the axoneme in a JhylacZ/lacZ brain.Magnification for all TEM images is 100,000" . Corresponding regions of the lateral ventricle were examined in each mouse (n#3 each for Jhy+/+ and JhylacZ/lacZ), andrepresentative images were taken with scale bars embedded at 75" ((A) and (B)), 2000" ((C) and (D)), and 8000" ((E) and (F)).

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point (Fig. 5B). Ependymal cilia develop along the lateral ventricularwalls beginning at birth in a caudorostral–ventrodorsal direction.In Jhy+/+ animals, large portions of the caudal ventricular walls arecovered with ciliary tufts, while the cilia are less dense across morerostral areas (Fig. 5A). The cilia display a consistent directionalorientation suggestive of the coordinated motion required for laminarflow (Fig. 5C and E). In P5 JhylacZ/lacZ animals, cilia development hasprogressed equally far across the lateral ventricular wall, but at lowmagnification the cilia appear less dense than in comparable ventri-cular regions of Jhy+/+ animals (Fig. 5B and D). At higher magnification,the cilia of JhylacZ/lacZ animals are sparser, shorter and of irregularlength, and oriented in random directions, suggesting a loss ofcoordinated movement (Fig. 5F). Similar data were obtained from allregions analyzed across the ciliated areas of the ventricles, regardlessof the density of the developing cilia at each region.

Transmission electron microscopy (TEM) was used to analyzethe ultrastructure of JhylacZ/lacZ lateral ventricular ependymal ciliain P5 brains. Normal motile cilia display a 9+2 arrangement ofmicrotubule doublets, and this pattern was observed in 99% of theJhy+/+ ependymal cilia (Figs. 5G and 8D). In JhylacZ/lacZ mice, only 7%of cilia displayed the normal 9+2 pattern, with 71% of cilia havinga 9+0 microtubule arrangement, and 19% having an 8+2 pattern(Figs. 5H, I and 8D). The microtubule pairs found in the center ofJhylacZ/lacZ 8+2 cilia do not display the characteristic singletmorphology of the central pair of normal 9+2 cilia. Rather, theyshow the asymmetric A/B structure of a peripheral doublet thathas moved to the center (Fig. 5I). In some axonemes this aberrantdoublet is found positioned between the center of the axonemeand an open position in the outer ring (Fig. 5J). Although furtherinvestigation is needed, this pattern suggests a 9+0 ciliary

axoneme in the process of reorganizing to form an 8+2 axoneme.All other axonemal components visualized, including the dyneinarms and radial spokes, appeared normal (Fig. 5H and I).

The JhylacZ transgene integrated into an uncharacterized gene,4931429I11Rik

Enhancer trapping with a reporter gene allows the bacterially-derived lacZ gene to be used as a tag to identify the transgeneintegration site. The GenomeWalker Universal Kit was used toidentify the integration site of the JhylacZ transgene. An adaptor-tagged restriction library was generated from JhylacZ/lacZ genomicDNA, which served as a template for PCR using primers within thetransgene against primers corresponding to the adaptors. Ampli-fication yielded a junction fragment containing the 5! end of theJhylacZ transgene, along with endogenous flanking sequence thatplaced the integration on mouse chromosome 9 at 40.9 Mb, withinexon 5 of the uncharacterized gene 4931429I11Rik (Fig. 6A and B).No function has been reported for this gene, and therefore thename Jhy has been approved by the Mouse Nomenclature Com-mittee to reflect its presumed role in the JhylacZ/lacZ phenotype. TheJhylacZ transgene integrated at the beginning of Jhy exon 5, in thesame orientation as the gene (Fig. 6B). To characterize theintegration boundary of the 3! end of the transgene, a series ofprimers were designed within the Jhy gene flanking the integra-tion site. Detailed PCR analysis determined that the integrationgenerated a 513 bp deletion that removes 425 bp of intronicsequence plus the exon 5 splice acceptor site and the first 88 bpof Jhy exon 5 (Fig. 6B and C).

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Fig. 6. The JhylacZ transgene disrupts the uncharacterized gene 4931429I11Rik (Jhy). (A) The JhylacZ transgene integrated within the Jhy gene on mouse chromosome 9. Boxesdenote the Jhy gene and flanking genes Bsx and Crtam, the arrowhead indicates the JhylacZ integration, the arrows indicate the direction of transcription and the primers usedfor qRT-PCR are denoted with half-arrows. (B) The JhylacZ transgene integrated at the beginning of Jhy exon 5, generating a deletion that removes 425 bp of intron 4 and thefirst 88 bp of exon 5. The black boxes represent Jhy exons, the grey box the JhylacZ transgene, the dotted line the intronic sequence deleted, and the bracket indicates theentire deletion. (In this view the chromosomal orientation of the Jhy gene has been rotated for clarity.) (C) Jhy exons 4 and 5 are juxtaposed to show the exon 5 sequencedeleted. (D) qRT-PCR analysis of the expression levels of Jhy (white bars), Bsx (black bars), and Crtam (hatched bars) in P0.5 JhylacZ/+ and JhylacZ/lacZ brain in comparison to Jhy+/+ levels (n≥7 for all genotypes), n denotes p≤0.05.

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The Jhy gene is conserved across most vertebrate species, withall species analyzed carrying conserved syntenic regions alsoencompassing Bsx and Crtam. At the nucleotide level, the mouseJhy cDNA has 72% identity to the human cDNA, 56% to the chicken,and 52% to the zebrafish. In the mouse, Jhy has nine exons thatwould generate an mRNA of 3068 nucleotides, and encode aprotein of 770 amino acids with a molecular weight of 87 kDa.At the protein level, conservation between mouse and human is66%, between mouse and chicken 27%, and between mouse andzebrafish 24% (Supplementary Fig. S1). Analysis of the predictedJHY protein revealed no known functional domains, and beyondthe single ortholog in all vertebrates there are no obvious paralogsin any species. The biological role of Jhy is therefore still to bedetermined.

Jhy mRNA and protein are reduced in JhylacZ/lacZ mice

The autosomal recessive inheritance pattern of the JhylacZ/lacZ

phenotype suggests that the integration generated a loss of functionor hypomorphic allele of Jhy. Quantitative RT-PCR (qRT-PCR) was usedto analyze Jhy expression levels in the brains of Jhy+/+, JhylacZ/+ andJhylacZ/lacZ mice at P0.5. Jhy expression levels were reduced to 85% ofwild type in JhylacZ/+ brains and to 41% in JhylacZ/lacZ brains (Fig. 6D). Theprimers used for this analysis span Jhy exons 2–3, detecting alltranscripts containing the 5! end of the Jhy gene. The transcriptionaltermination signal of the JhylacZ transgene is expected to disrupt alltranscripts at the exon 5 integration point, but the actual transcriptsproduced from this allele are currently unknown. Should transcriptionproceed beyond the transgene integration site, the deletion of the first88 bp of exon 5 means that a fully wild type transcript cannot beproduced.

The Brain specific homeobox (Bsx) and Cytotoxic and regulatory T cellmolecule (Crtam) genes are closely linked to Jhy, with Bsx located 17 kbproximal and Crtam located 9 kb distal (Fig. 6A). Transgene integra-tions can cause regional changes in gene expression, and thereforeexpression of Bsx and Crtam were similarly examined by qRT-PCR inP0.5 brains. Levels of Crtamwere not significantly altered in JhylacZ/+ orJhylacz/lacZ brains, but Bsx levels were reduced to 77% of wild type inJhylacZ/+ and to 67% in Jhylacz/lacZ (Fig. 6D). These data suggest a long-range effect of the JhylacZ transgene, and potential functional linkage ofJhy and Bsx expression.

To explore the levels of JHY protein in JhylacZ mice, a commer-cial antibody generated against the human JHY homolog(C11ORF63) was used (Sigma-Aldrich, St. Louis, MO) (Ivliev et al.,2012). Ivliev et al. (2012) carried out a bioinformatics screen forproteins potentially expressed in ciliated cells, identifyingC11ORF63 among 74 novel predicted ciliary proteins. Ciliaryexpression of these proteins was verified with data from theHuman Protein Atlas, a high-throughput analysis of protein

expression data across human tissues (Uhlen et al., 2010). Thisstudy had explored C11ORF63 expression in several ciliated tissuesusing a peptide antibody directed against the exon 3-encodedregion of the predicted C11ORF63 protein. This antibody wasobtained and tested against P5 mouse brain, and found torecognize the mouse JHY protein as well (Fig. 7A). This was notsurprising, as the C11ORF63 peptide antigen bears 87% identity tothe homologous mouse sequence. In Jhy+/+ P5 mouse brainsections, expression of JHY was seen in the ependymal cells liningthe lateral ventricles (Fig. 7A), and scattered cells within deeperlayers, but this protein signal was significantly reduced inJhylacz/lacZ animals (Fig. 7B).

The JhylacZ phenotype can be rescued by a Jhy transgene

The most definitive way to verify that a specific gene underliesa particular phenotype is to cure the phenotype by restoring thecandidate gene. Bacterial artificial chromosomes (BACs) are idealfor transgenic rescue, since their large size makes it possible toisolate a gene and its regulatory elements on a single clone. Rescueof the JhylacZ phenotype was performed using the BAC clone RP23-273J15 that spans the region from 105 kb proximal to 65 kb distalto the JhylacZ integration site (Fig. 8A) (http://bacpac.chori.org). TheRPCI-23 library is derived from the C57BL/6 mouse strain, allowingthe use of strain-specific polymorphisms to distinguish the endo-genous Jhy gene from the BAC Jhy gene, and endogenous fromBAC-derived transcripts. The BAC clone was linearized and micro-injected into FVB/N embryos and a single transgenic mouse linewas obtained that both transmitted and expressed the BAC Jhygene. Formally named Tg(RP23-273J15)1Jvs, this line is abbreviatedhere as TgBAC. The transgene was crossed into the JhylacZ line togenerate animals heterozygous for TgBAC and homozygous forJhylacZ (TgBAC/+;JhylacZ/lacZ).

Analysis of these mice revealed that a single copy of TgBAC yielded a50% rescue of JhylacZ/lacZ mice, with the incidence of hydrocephalusreduced from 74% in JhylacZ/lacZ mice to 37% in TgBAC/+;JhylacZ/lacZ mice(Fig. 8B). Phenotypically, TgBAC/+;JhylacZ/lacZ animals displayed reduceddoming of the skull, though subtle changes were still observed insome animals (Fig. 8C). Further analysis of the TgBAC integration,however, revealed that the BAC transgene had been truncated withinthe Jhy gene, likely during integration into the genome. Directsequencing of PCR products across regions containing single nucleo-tide polymorphisms between FVB/N (JhylacZ background) and C57BL/6(TgBAC background) strains were used to determine that TgBAC wastruncated at a position between 419 bp into Jhy exon 5 and 2420 bpinto Jhy intron 5 (Fig. 8A). Interestingly, this point is just slightly 3! tothe position of the JhylacZ insertion in the endogenous Jhy gene.Between 60 and 70% of the Jhy protein coding sequence remains onTgBAC (Fig. 8A).

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Fig. 7. JhylacZ/lacZ mice express reduced levels of JHY protein. (A) JHY protein localizes to the ependymal cells of the lateral ventricle in Jhy+/+ P5 mouse brain using anantibody directed against the human JHY homolog C11ORF63. (B) JhylacZ/lacZ brain shows significantly reduced levels of JHY protein.

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Jhy expression in TgBAC/+;JhylacZ/lacZ P0.5 brains was assayed byqRT-PCR using the Jhy exon 2–3 primers and was found to average113% of wild type levels (data not shown). The TgBAC transgenetherefore restores the level of an, albeit truncated, Jhy mRNA. Bsx

expression, which is decreased to 77% of wild type in JhylacZ/lacZ

brains, was unchanged from this level in TgBAC/+;JhylacZ/lacZ mice;this is expected since Bsx is not included on the truncated TgBAC

(Fig. 8A). The increase in Jhy transcription conferred by the TgBAC

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Fig. 8. Transgenic rescue of JhylacZ/lacZ mice. (A) The RP23-273J15 BAC clone spans the region 80 kb proximal to 20 kb distal to the Jhy gene, while the integrated TgBAC hasbeen truncated to contain 60–70% of the protein coding region of Jhy. The white boxes represent the Bsx, Jhy and Crtam genes, the arrowhead indicates the JhylacZ integration,the arrows indicate the direction of gene transcription, the grey box indicates the end of the truncated portion of TgBAC that cannot be defined due to a lack of SNPs. (B) Theincidence of hydrocephalus is reduced from 74% in JhylacZ/lacZ mice (diamonds) to 37% in TgBAC/+; JhylacZ/lacZ mice (squares). (C) Image of JhylacZ/lacZ (left) and TgBAC/+; JhylacZ/lacZ

(right) littermates showing phenotypic rescue by TgBAC. (D) Quantification of TEM analysis of ependymal ciliary microtubule arrangement in Jhy+/+ (black bars), JhylacZ/lacZ

(white bars) and TgBAC/+; JhylacZ/lacZ (grey bars) at P5 (n#620 for Jhy+/+, 147 for JhylacZ/lacZ, and 117 for TgBAC/+; JhylacZ/lacZ), n denotes p≤0.05 relative to Jhy+/+, nn denotes p≤0.05relative to JhylacZ/lacZ.

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transgene was sufficient to enact partial rescue of the ciliaryphenotype as well; 32% of ependymal cilia displayed the normal9+2 microtubule configuration in TgBAC/+; JhylacZ/lacZ cilia, com-pared to only 7% of cilia in JhylacZ/lacZ (Fig. 8D). These resultssuggest that TgBAC produces a truncated JHY protein that retainspartial function.

Discussion

Jhy is a new candidate gene for hydrocephalus

A transgenic insertional mutation on mouse chromosome 9 ledto the development of juvenile hydrocephalus, and the subsequentdiscovery of the Jhy gene. None of the known murine hydroce-phalus mutations map to this genomic region, indicating thatJhylacZ is a new model for juvenile hydrocephalus. JhylacZ/lacZ micedevelop communicating hydrocephalus during early postnataldevelopment, with the earliest cases visible by histology at P1.5(Fig. 1 and data not shown). Grossly visible doming of the skull isoften observed by 2 weeks of age. These early onset hydrocephaluscases have died or must be sacrificed by 4–8 weeks of age (Fig. 2B).The onset and progression of the JhylacZ hydrocephalus varies,however, and a small percentage of animals instead develop a lateonset, milder form of the disease (Fig. 2C). These mice showsymptoms developing at 5–6 weeks of age, and the animalssurvive beyond a 12 week monitoring period.

The homozygous recessive inheritance pattern of the JhylacZ

phenotype suggests the disruption of a gene involved in CSFbalance, with reduction or loss of function of this gene causativefor the phenotype. Genomic analysis identified the locus disruptedby the transgene as carrying the uncharacterized gene4931429I11Rik, now named Jhy. The Jhy gene can be located in allwell-annotated vertebrate genomes, from zebrafish throughhumans. The expression pattern of the JhylacZ reporter genesuggests activity in the pineal gland, the hypothalamus, thechoroid plexus of the third ventricle, and the ependymal cellswithin the aqueduct of Sylvius. The ependymal cells and choroidplexus in particular play key roles in the production and flow ofCSF, suggesting altered expression in these tissues might underliethe JhylacZ/lacZ phenotype. While these experiments did not detectlacZ expression in the ependyma of the lateral ventricles, pre-liminary analysis of JHY protein by immunofluorescence indicatesthat JHY is present in these cells (Figs. 4 and 7).

Ciliary defects in JhylacZ/lacZ hydrocephalus

The most striking aspect of the JhylacZ/lacZ phenotype is thedisorganization and altered morphology of the ependymal cilia liningthe lateral ventricles (Fig. 5). By SEM, the ependymal cilia are sparser,shorter and show a loss of directional orientation that suggests theymay be nonfunctional (Fig. 5A–F). By TEM, only 7% of JhylacZ/lacZ ciliashow the normal 9+2 microtubule organization of motile cilia, withmost having instead a 9+0 or 8+2 configuration (Figs. 5G–J and 8D).Normal nonmotile cilia display a 9+0 confirmation, indicating that theJhylacZ/lacZ 9+0 cilia are almost certainly immotile, but little is knownabout the potential motility of 8+2 cilia. The unique central pairmorphology of 9+2 cilia is believed necessary for ciliary motility, withthe central pair controlling the direction of the stroke. The abnormaldoublet morphology of the central microtubules in the JhylacZ/lacZ 8+2cilia suggest these cilia may also be immotile. Loss of ciliary motilityleading to a failure to maintain CSF flow is therefore the predictedmechanism underling the JhylacZ/lacZ hydrocephalus. The JHY protein isnot found in the ciliary proteome database (www.ciliome.com), but arecent bioinformatic-based study identified the predicted protein of

the human homolog of Jhy (C11ORF63) in the cilia of adult human lungand oviduct (Ivliev et al., 2012).

Studies involving mouse models of hydrocephalus show a require-ment for proper cilia structure and function in regulating CSF flow.Only two existing hydrocephalic models, however, show disruption ofciliary microtubule patterning. Hsf1 (Heat shock transcription factor 1)-null mice display reduced ciliary beat amplitude and frequency, anddevelop chronic sinusitis and communicating hydrocephalus (Takakiet al., 2007). Ultrastructural studies of Hsf1 respiratory epitheliumshowed that approximately 10% of cilia were structurally abnormal,with deletion and/or disorganization of the central pair and outerdoublet microtubules. The abnormal cilia development in Hsf1mutants was predicted to result from a secondary decrease in Hsp90(Heat shock protein 90), which facilitates tubulin stability and poly-merization (Takaki et al., 2007). Mice mutant for the Stumpy genedevelop hydrocephalus and polycystic kidney disease (Town et al.,2008). The ependymal cells of Stumpy mutant animals contain intactbasal bodies, but lack ciliary axonemes entirely. Localization ofSTUMPY protein within the axonemes suggests it may be involvedin the intraflagellar transport of proteins required for cilia outgrowth.

Regional gene expression is altered, and JHY protein is lost,in JhylacZ/lacZ mice

JhylacZ/+ mice expressing 85% of the wild type level of Jhy do notdevelop hydrocephalus, while JhylacZ/lacZ mice expressing 41% ofthe wild type level develop hydrocephalus with a penetrance of74%. These numbers suggest a threshold for Jhy expression, wherethe development of hydrocephalus occurs between 41 and 85% ofwild type Jhy expression. The incomplete penetrance of the JhylacZ/lacZ hydrocephalus may therefore be due to stochastic events thatsubtly alter Jhy expression levels in JhylacZ/lacZ mice. In consideringexpression from the JhylacZ allele, however, it is important toconsider the transcripts that could be produced. The integrationof a transcriptional termination codon suggests that JhylacZ tran-scripts should contain at most the first 34% of the wild typetranscript, which would encode 42% of the full length protein.Deletion of the first 88 bp of exon 5 dictate that any transcriptproduced beyond the integration site cannot be wild type innature. The most likely mechanism for reconstructing a near-fulllength transcript, splicing from exon 4 to exon 6, would generatean early stop codon. An antibody generated against the human JHYhomolog C11ORF63 found JHY protein localized to the ventricularependymal cells of Jhy+/+ P5 mouse brains, while sections of JhylacZ/lacZ brains showed a significant reduction in JHY protein (Fig. 7).These data support a Jhy loss of function mechanism for the JhylacZ

mutation. Future studies will further characterize the proteinproducts made from both Jhy+/+ and JhylacZ alleles.

Restoring a truncated version of the Jhy gene, roughly the sameportion of the transcript as lies upstream of the JhylacZ integration,provides significant rescue of the hydrocephalus phenotype (Fig. 8).TgBAC/+;JhylacZ/lacZ animals show near-wild type levels of Jhy transcriptwhen analyzed using primers spanning exon 2–3, and the phenotypicrescue suggests the mechanism is likely to be increased levels of atruncated protein. The JHY protein may therefore be modular innature, where key domains within the remaining portion of theprotein can provide partial function. There is some precedence forthe rescue of ciliary defects, particularly structural defects, with genefragments in other systems. A construct expressing just 35% of theChlamydomonas centriolar protein Bld10p gave partial rescue of theBld10p deletion phenotype (Hiraki et al., 2007). Bld10p is a componentof the radial spokes of the centriolar cartwheel, and the truncatedprotein allowed the assembly of largely normal flagella, but withshorter spokes and eight rather than the expected nine microtubuletriplets.

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Please cite this article as: Appelbe, O.K., et al., Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning andjuvenile hydrocephalus. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.003i

Expression analysis showed decreased levels of Bsx in JhylacZ/lacZ

brains, and no change in the expression of Crtam. Interestingly, theBsx expression pattern overlaps with that predicted for Jhy inpineal gland and hypothalamus, though not in choroid plexus orependyma (McArthur and Ohtoshi, 2007). There is no demon-strated role for Bsx in CSF balance or the development of hydro-cephalus, and mouse models carrying a deletion of the Bsx geneare fully viable but display altered feeding and nursing behaviors(McArthur and Ohtoshi, 2007; Sakkou et al., 2007). Crtam levelsare not altered in JhylacZ/lacZ mice, and deletion of this gene displaysno neural phenotype (Yeh et al., 2008). Taken together, theseexpression and rescue data provide strong support for alteredfunction of the Jhy gene as causative for the JhylacZ/lacZ hydro-cephalus. It remains formally possible that altered expression ofBsx may contribute in part to the JhylacZ/lacZ phenotype, and itsabsence from TgBAC may underlie the lack of full rescue. Ongoingexperiments will generate a targeted deletion of the Jhy gene,along with additional TgBAC animals, to more clearly define allcontributory factors.

Ependymal- and choroid plexus-based models for JhylacZ

hydrocephalus

At least two models can be proposed for the mechanism of theJhylacZ hydrocephalus. First, loss of ciliary-mediated CSF flow maycause fluid accumulation in the ventricles resulting in hydroce-phalus. Jhy appears to function in the ventricular ependymal cellsto regulate cilia development, with loss of Jhy causing abnormallypatterned ciliary microtubules. Hydrocephalus in the JhylacZ micearises as early as P1.5, suggesting the initiating event must precedethis age. Previous analysis has shown that the ependymal cilia ofthe lateral ventricles develop between birth and P10, while thecilia lining the aqueduct of Sylvius are already present by P1(Banizs et al., 2005; Spassky et al., 2005; Tissir et al., 2010). Ourexperiments find abundant cilia in the caudal region of the Jhy+/+

lateral ventricles by P5, and these cilia are abnormal in JhylacZ/lacZ

mice. JHY protein is present in the ependymal cells of the lateralventricles at P5, and expression of the JhylacZ reporter suggestsexpression in the ependymal cells lining the aqueduct at P0.5(Figs. 4R and 7). These data are consistent with loss of ciliaryfunction in the aqueductal and/or ventricular ependyma beingcausative for JhylacZ hydrocephalus.

Expression from the JhylacZ transgene is also found in thechoroid plexus of the third ventricle, yet histological analysis ofthe choroid plexus by light microscopy shows no obvious abnorm-alities (Figs. 3D and 4L and data not shown). The choroid plexusepithelium carries motile cilia that develop by P1, though thesecilia contribute minimally to bulk CSF flow and instead appear tofunction in a signaling pathway that regulates CSF production(Banizs et al., 2005). In the Tg737orpk mutant mouse, loss of normalcilia function on the choroid plexus leads to excessive CSFproduction and hydrocephalus (Banizs et al., 2005). Abnormalchoroid plexus cilia might therefore initiate hydrocephalusthrough CSF oversecretion, with loss of ventricular ependymalflow exacerbating the increased fluid load. Further investigation ofthe choroid plexus cilia will determine their role in the develop-ment of hydrocephalus in JhylacZ/lacZ mice.

Acknowledgements

The authors thank Lydia Johns, Jonathan River, Greg Karczmarand Marta Zamora at the University of Chicago MRIS Core Facilityfor assistance with MRI imaging, Kathleen Millen at the Universityof Washington for interpretation of mouse brain pathology, JackGibbons for assistance with electron microscopy, Ekaterina

Steshina and Elena Glick for technical assistance in the earlyphases of this work, and members of the Schmidt lab forcomments on the manuscript.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ydbio.2013.07.003.

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