a novel murine allele of intraflagellar transport protein 172 causes

A novel murine allele of Intraflagellar TransportProtein 172 causes a syndrome includingVACTERL-like features with hydrocephalus

Joshua M. Friedland-Little1,2, Andrew D. Hoffmann1,2, Polloneal Jymmiel R. Ocbina3,

Mike A. Peterson1,2, Joshua D. Bosman1,2, Yan Chen4, Steven Y. Cheng4,

Kathryn V. Anderson3 and Ivan P. Moskowitz1,2,∗

1Department of Pediatrics and 2Department of Pathology, The University of Chicago, Chicago, IL 60637, USA,3Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA and 4Department of

Developmental Genetics, Nanjing Medical University, Nanjing, Jiangsu 210029, China

Received January 21, 2011; Revised April 29, 2011; Accepted May 25, 2011

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate developmentand human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERLassociation with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intrafla-gellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically,avc1 caused VACTERL-H but not abnormalities in left–right (L–R) axis formation. Avc1 resulted in structuralcilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement forIft172 in ciliogenesis using an allelic series generated with Ift172avc1 and Ift172wim, an Ift172 null allele:cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrastto >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific ciliumpopulations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% ofnormal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1embryos, with Hh signaling abnormalities but not L–R abnormalities, had cilia equally divided betweenfull-length and truncated, and avc1/wim embryos, with both Hh signaling and L–R abnormalities, were pri-marily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including dis-rupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation ofIft172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L–Raxis determination, and resulting in the VACTERL-H phenotype.

INTRODUCTION

VACTERL association is a non-random association of conge-nital anomalies with no known etiology. First described asVATER association by Quan and Smith in 1973, the initialpatients were noted to have vertebral anomalies (V), analatresia (A), tracheoesophageal fistulas (TE) and renal (R)and limb (L) anomalies. The high incidence of congenitalheart defects in patients with VATER association was sub-sequently described, resulting in the inclusive acronymVACTERL (1–3).

The etiology of VACTERL association has not been estab-lished. Currently, patients can be diagnosed with VACTERL

association if they have two or more of the relevant anomalies

(4). In the series reported by Khoury et al. (5), .90% of

patients diagnosed with VACTERL association had three or

fewer anomalies, and ,1% of patients had all six anomalies.

There have been few reported instances of familial recurrence

of typical VACTERL, and therefore monogenic causation has

been deemed unlikely. Interestingly, Mendelian inheritance of

a distinct subgroup of VACTERL association, VACTERL

∗To whom correspondence should be addressed at: Departments of Pediatrics and Pathology, The University of Chicago, 900 East 57th Street, KCBDRoom 5118, Chicago, IL 60637, USA. Tel: +1 7738340462; Fax: +1 7738342132; Email: [email protected]

# The Author 2011. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2011, Vol. 20, No. 19 3725–3737doi:10.1093/hmg/ddr241Advance Access published on June 8, 2011

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with hydrocephalus, or VACTERL-H, has been reported (6).Patients with VACTERL-H meet the diagnostic criteria forVACTERL, and in addition have hydrocephalus. Both autoso-mal recessive and X-linked recessive inheritance ofVACTERL-H have been described ((7,8) reviewed in 9).These inheritance patterns have led some authors to arguethat VACTERL-H should be classified as a syndrome (asopposed to an association) despite the unknown genetic etiol-ogy, given the evidence for monogenic inheritance (10).

Mice with disrupted Hedgehog (Hh) signaling display phe-notypic overlap with VACTERL patients. In particular, Gli2and Gli3 mutant mice have vertebral anomalies (11); Shhmutants develop truncated limbs (12); Gli3 mutants exhibitpolydactyly (13); Shh, Gli2 and Gli3 mutants all showvarious foregut and hindgut anomalies (14–17); and Shh andGli2;Gli3 double-mutants show renal anomalies (18–20).These findings have led to speculation that alterations of theHh signaling pathway may contribute to the pathogenesis ofhuman VACTERL (4). Recently, a human patient withtypical VACTERL association was found to have a mutationof HOXD13 (21), a downstream target of Shh, providing poss-ible indirect evidence linking human VACTERL with the Hhsignaling pathway. However, no models of VACTERL-H haveidentified a single-gene etiology to date.

A growing body of work has demonstrated the crucial roleplayed by primary cilia in vertebrate developmental signalingpathways. Multiple studies have begun to elucidate thecomplex role played by cilia and intraflagellar transport in Hhsignaling. Huangfu et al. (22) first made the connectionbetween cilia and Hh signaling, demonstrating that wimple(wim), a null allele of intraflagellar transport protein 172(Ift172), results in absent cilia and loss of Hh signaling. Sub-sequently, it has been established that multiple Hh signaling mol-ecules, including Ptch1, Smo, Sufu and the Gli family oftranscription factors, are all enriched in cilia (23–27). Intrafla-gellar transport appears to play an active role in Hh signaling,and IFT proteins are required for both Gli activator and Glirepressor function (25,28,29). Therefore, cilia mutations causephenotypes consistent with loss of Hh signaling in some develop-mental contexts and gain of Hh signaling in others. The preciseciliary structural requirements for Hh signaling remain elusive.

Although cilia function has been implicated in both Hh sig-naling and in left/right axis determination, mechanisms dis-tinguishing these two crucial cilia functions have not beenestablished. Situs abnormalities are a common feature of pre-viously described ciliary mutations. Mutations in ciliogenesisgenes that completely eliminate cilia impair both functions,making investigation of the separate requirement for intracili-ary molecular signaling from the mechanical role of the ciliain this process unclear. The primary ciliary dyskinesia pheno-type, with situs anomalies but no other apparent Hh-relateddefects, suggests that immotile cilia are unable to create thenecessary nodal flow required for L–R determination, butcan otherwise carry out signal transduction normally. Conver-sely, the Oak Ridge polycystic kidney (ORPK) mouse, homo-zygous for partial loss-of-function of Ift88, demonstratedintact left–right (L–R) axis determination but evidence ofabnormal Hh signaling (30), suggesting that it is possible todisrupt Hh signaling while maintaining the ciliary functionsneeded for situs determination.

Here we report a mouse model for VACTERL-H identified ina forward genetic screen for congenital heart disease (CHD) inmice (31). The recessive N-ethyl,N-nitrosurea (ENU)-inducedmutation, avc1 (atrioventricular canal 1), caused theVACTERL-H phenotype including vertebral anomalies, analatresia, cardiac defects, tracheoesophageal anomalies, renaldysplasia, limb anomalies and hydrocephalous. We identifiedan avc1-specific polymorphism in Ift172, an intraflagellar trans-port gene previously implicated in the Hh signaling pathway(22). Avc1 is a hypomorphic allele of Ift172, causing abnormalsplicing and decay of the majority of Ift172 transcript. Heartdefects in avc1 mutant mice result from the specific loss of anHh-responsive lineage that generates the atrial septum of theheart, providing the first molecular link between a cilia mutationand CHD. Although avc1 caused significant truncation of nodalcilia, no left/right axis abnormalities were observed in mutantembryos. Using Ift172avc1 and Ift172wim, we generated anIft172 allelic series to investigate the relationship betweenIft172 dosage and cilia architecture, IFT and Hh signaling. Weobserved that dose reduction of Ift172 caused anomalies ofnodal cilia in vivo, primary cilia in vitro and a disruption ofHh signaling in a dose-dependent manner. Truncated mutantcilia displayed both abnormal localization of IFT88 and Gli2to the distal ciliary tip, suggesting a defect of the distal ciliaryaxoneme. Our findings implicated structural cilia defects inthe pathogenesis of VACTERL and suggested that cilia genes,and in particular IFT genes, are likely candidates for disease-causing mutations in VACTERL-H.

RESULTS

avc1: an ENU-derived mouse mutant with phenotypicfeatures of VACTERL-H

We performed a forward genetic screen for ENU-inducedmutations that caused perinatal lethality and CHD in themouse (31). avc1 caused heritable syndromic atrioventricularseptal defects (AVSDs) as part of VACTERL-H association(Fig. 1). avc1 mutant newborns demonstrated hydrocephaluswith highly penetrant vertebral, anal, cardiac, limb andpalate abnormalities (Fig. 1U). Anomalies of the cervical ver-tebrae, including absent spinal processes or abnormallyformed transverse processes, were present in 8/10 avc1mutant mice in contrast to 0/10 wild-type littermate controls(P ¼ 0.02; Fig. 1A and B). Mutants had seven cervical ver-tebrae, as is standard, but each cervical vertebra lacked thevertebral body (Fig. 1B). Thoracic, lumbar and sacral ver-tebrae appeared normal. Anal atresia, evidenced by disconti-nuity between the anal squamous epithelium and rectalcolumnar epithelium, was present in three of five avc1embryos, whereas all the eight wild-type embryos showed aclear, intact transition between anal and rectal epithelium(P ¼ 0.04; Fig. 1C and D). Cardiac anomalies, atrioventricularseptal defects with common atrium, were present in 100% ofavc1 mutants analyzed (n . 100; P , 1026; Fig. 1E and F).Cardiac defects were not observed in any littermate controlanimals. Bone staining demonstrated anomalies of the longbones of the forelimbs and hindlimbs in mutant embryos(Fig. 1K–N). Mutant embryos demonstrated shortening ofthe humerus, radius, ulna, femur, tibia and fibula. In addition,

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all mutant mice demonstrated preaxial polydactyly of both theforelimbs and hindlimbs (n ¼ 119) (Fig. 1N). All newbornanimals examined demonstrated abnormal facies, with short-ening of the muzzle. Clefts of the secondary palate were simi-larly noted in 20/20 newborn mutants (P , 0.01) (Fig. 1P).Hydrocephalus was present in four of five mutant animals,but none of seven litter mate wild-type controls (P ¼ 0.01;Fig. 1Q and R).

The remaining features of the VACTERL-H phenotype,tracheo-esophageal and renal abnormalities, were present

with variable frequency. Esophageal and tracheal anomalieswere observed in 2/9 mutant embryos at E14.5 (esophagealstenosis with enlarged, cystic trachea in one case, and esopha-geal atresia in the other), whereas 0/10 wild-type embryos ana-lyzed showed foregut anomalies (P ¼ 0.1; Fig. 1G and H).Renal dysplasia with hypoplastic glomeruli was observed infour of nine avc1 mutant embryos compared with none ofsix wild-type (P ¼ 0.09, Fig. 1I and J). No eye abnormalitieswere observed by gross or histologic analysis of avc1mutant embryos (n ¼ 5).

Figure 1. Avc1 mutant mice have VACTERL association with hydrocephalus. (A and B) Bone stain demonstrated normal cervical vertebral anatomy in newborncontrol animal (A), whereas mutant littermate exhibited the absence of vertebral bodies (B). Bone is stained red and cartilage blue. (C and D) Sagital sectionthrough an E14.5 control embryo demonstrated anorectal epithelial continuity with normal anatomy (C). Corresponding section through mutant littermateshowed anal atresia with discontinuity between the squamous epithelium of the anus (small arrow, D) and the columnar epithelium of the distal rectum(large arrow, D). (E and F) Transverse section through the heart of the E14.5 control embryo showed intact atrial and ventricular septation (E). Correspondingsection through the mutant littermate heart showed an atrioventricular septal defect (asterisk) with the common atrium. (G and H) Transverse section of theE14.5 control embryo demonstrated normal foregut anatomy (G). Mutant littermate (H) displayed a hypoplastic esophagus (Es) with an enlarged and abnormaltrachea (Tr). (I and J) Transverse section of the E14.5 control embryo showed normal renal histology (I). Corresponding section through the mutant littermate (J)demonstrated dysplasia with hypoplastic glomeruli (arrow). (K–N) Bone stain of the newborn control animal showed normal bones of forelimb and hindlimb (Kand M). Mutants exhibited shortening of the long bones of both fore- and hindlimbs (L and N) and preaxial polydactyly in the forelimb (inset, N). (O and P)Gross specimen of newborn wild-type palate (O). Mutants exhibited a cleft of the secondary palate (arrow, P). (Q and R) Sagital section through the head of thenewborn avc1 mutant showed ventriculomegaly compared with the littermate control animal of normal ventricular size (Q). (S and T) Gross external morphologyof E12.5 avc1 embryo demonstrated abnormal facies with a shortened muzzle (arrow, T) compared with the littermate control of normal size. (U) Table ofprevalence of VACTERL-H features in avc1 mutants and littermate controls.

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Interestingly, no cardiac looping anomalies were noted inany of the mutant embryos. All mutant embryos analyzed(n . 100) showed levocardia with normal, d-looping of theventricles, as did all wild-type control embryos. Similarly,all mutant embryos demonstrated situs solitus of the abdomi-nal organs. Thus, no situs anomalies were observed in avc1mutant embryos.

avc1 is an allele of Ift172

A candidate gene approach was used to identify the avc1mutation. Recombinant chromosomes were identified inaffected animals from an intercross between known carriersthat located the avc1 mutation between markers D5MIT334and D5MIT419. The interval containing avc1 corresponded

to 0.9 megabases (Mb) on the physical map containing 33genes. Bioinformatic investigation of the 33 candidate genesrevealed no known role for any in heart development.However, one gene in the interval, Ift172, encoded an intrafla-gellar transport protein required for cilia biogenesis and impli-cated in the Hh signaling pathway (22).

Sequencing of all 48 exons of the Ift172 gene, includingadjacent intronic sequence, revealed an A-to-G transition inthe splice donor site downstream of exon 24 (Fig. 2A). Thispolymorphism, located at position +3 in the 3′ intron ofexon 24, altered a nucleotide conserved from humans toopossum. We hypothesized that the splice-site sequencealteration may affect the splicing of Ift172 transcripts. Weevaluated products of Ift172 transcription by reverse transcrip-tase polymerase chain reaction (RT-PCR) in wild-type and

Figure 2. Aberrant splicing of Ift172 in Avc1 mutant embryos. (A) Sequencing of Ift172 in Avc1 mutants demonstrated an A-to-G transition at position +3 ofexon 24 splice donor. Mis-splicing and exon 24 skipping resulted in theoretical transcription encountering a stop site in exon 25. (B) RT-PCR spanning Ift172exons 22 to 26 from three wild-type (left lanes) and three Avc1 mutant (right lanes) E10.5 embryos. Multiple splice products were obtained from Avc1 but notwild-type embryos. (C) Sequence tracings from gel-purified RT-PCR products shown in (B). RT product from wild-type embryos (I) and minority product fromAvc1 embryos (II) demonstrated wild-type cDNA sequence. The majority product from the Avc1 embryos (III) demonstrated exon 24 skipping. (D) Avc1 failed tocomplement Ift172wim. Embryos with either the avc1 or Ift172wim alleles appeared normal. Embryos carrying both alleles were all affected.



avc1 mutant embryos, using primers designed to amplifyIft172 transcriptional products generated between exons 22and 26, spanning the exons adjacent to the sequence poly-morphism. Semi-quantitative RT-PCR from wild-type whole-embryonic tissue at E10.5 revealed a single product, whosesequence corresponded to wild-type Ift172 cDNA (Fig. 2Band C). RT-PCR from avc1 mutant whole-embryonic tissueat E10.5 revealed two distinct products. The sequence of theminority species, of higher molecular weight, correspondedto wild-type Ift172 cDNA. However, the sequence of the

majority species, of lower molecular weight, revealed exon24 skipping (Fig. 2B and C). Theoretical translation of themis-spliced message encountered a frame shift and prematuretermination in exon 25 (Fig. 2A).

The results of RT-PCR from avc1 mutant embryossuggested that the sequence alteration identified in Ift172had functional consequences on Ift172 splicing. However,this perturbation of Ift172 had yet to be directly linked tothe phenotype observed in avc1 mutant embryos. Becausethe avc1 allele was generated by ENU mutagenesis, itremained feasible that an undiscovered mutation affectinganother gene in the physical interval containing avc1 contrib-uted to the mutant phenotype. We sought to directly testwhether avc1 and Ift172 were allelic. We performed a comple-mentation test between avc1 and Ift172wim, an Ift172 nullallele that causes mid-gestation embryonic lethality and lossof Hh signaling (22). The embryos from an intercrossbetween Ift172wim/+ and avc1/+ mice were evaluated. Micecarrying both the Ift172wim and avc1 alleles demonstratedcardiac, limb and craniofacial abnormalities, whereas noneof the heterozygous carriers of either mutant allele demon-strated phenotypic abnormalities (Fig. 2D). These findingsdemonstrated that Ift172wim and avc1 failed to complementand provided further evidence that the identified mutation ofthe Ift172 gene is responsible for the phenotype observed inavc1 mutant mice. As Ift172avc1 mice maintained some wild-type transcript and die at birth whereas Ift172wim mice die atE10.5, we conclude that avc1 is a hypomorphic allele. Nohomozygous avc1 mice were observed at weaning from 200consecutive litters.

Whole-mount in situ hybridization against Ift172 in controlembryos showed widespread expression of Ift172 in tissuesaffected by avc1 and known to be Hh-responsive (Fig. 3A–C, left column). Ift172avc1 mutant embryos demonstratedgreatly reduced but detectable expression (Fig. 3A–C, rightcolumn). These observations suggested that the majorityproduct of mis-spliced mRNA in the mutant embryos under-went nonsense-mediated decay. To directly test this hypoth-esis, we performed western blotting with an antibody againstthe N-terminus of IFT172 from E10.5 embryonic extracts.avc1 mutant embryos expressed 18% of the amount ofIFT172 compared with littermate wild-type controls after nor-malization (Fig. 3D). Heterozygous avc1 mutant embryosexpressed 63% of the amount of IFT172 compared with litter-mate wild-type controls after normalization (Fig. 3D). Theseresults provide additional evidence that the mis-splicedproduct of Ift172 RNA undergoes nonsense-mediated decayin avc1 mutant embryos, and that avc1 causes a dose decreasein IFT172 protein levels.

Ift172 mutant embryos have defects in Hh signaling

Given the known role of Ift172 in Hh signaling and the sus-pected link between defective Hh signaling and VACTERLsyndrome, we sought to evaluate the impact of reducedIft172 expression on Hh signaling in Ift172avc1 mutant mice.We assessed the integrity of the Hh signaling pathway inIft172avc1 mutants in two ways, by whole-mount analysis ofan Hh reporter and biochemically. First, we qualitatively eval-uated the expression pattern of Patched1 (Ptch1) and Gli1.

Figure 3. Reduced expression of Ift172 in avc1 mutant embryos. (A–C)Whole-mount in situ hybridization against Ift172 at E8 (A), E9 (B) and E10(C) showed widespread expression of Ift172 in tissues with known require-ments for Hh-signaling in control embryos (left column) with minimalexpression in the heart. Ift172 mRNA was barely detectable in mutant litter-mates at each time point, with faint staining primarily along the neural tube(A–C, right column). (D) IFT172 protein was quantitatively determined bywestern blot analysis using a rabbit antibody raised against the N-terminusof IFT172. Lanes represent whole-embryo extracts from wild-type (lane 1),avc1/+ heterozygote (lane 2) or avc1/avc1 mutant (lane 3) embryos atE10.5, with g-tubulin used as a loading control. The filled arrowhead indicateswild-type IFT172 protein and the two asterisks indicate major non-specificbands at �65 and 105 kDa. IFT172 band intensities normalized tog-tubulin: wild-type ¼ 1.00 (+0.14 SD), avc1/+ ¼ 0.6297 and avc1/avc1 ¼ 0.1826.



The expression of Ptch1, the primary Hh membrane receptor,and Gli1, a member of the Gli transcription factor familyresponsible for transducing the Hh signal, are Hh dependent.We compared the expression of Ptch1 using a previouslyreported Ptch1-lacZ Hh-reporter mouse line (32) and Gli1by in situ hybridization in Ift172avc1 mutants and wild-type lit-termate controls at E10.5 (Fig. 4). In wild-type embryos, Ptch1and Gli1 expression were confined to regions with known Hhsignaling mirroring the expression of Ift172 (Fig. 4A). In com-parison, Ift172avc1 mutant littermates demonstrated a globalreduction in the expression of Ptch1 and Gli1 (Fig. 4B). Con-sistent with previous analysis describing an extra-cardiacpopulation of Hh-receiving progenitor cells that subsequentlymigrate into the heart to form the atrial septum (33), therewas minimal evidence of Hh signaling within the heart itself(Fig. 4A and B, lower panels).

We next quantitatively assessed Hh signaling in Ift172avc1

embryos, examining the proteolytic processing of the Gli3transcription factor. In mammals, Gli3 exists in two forms,a full-length protein that acts as a transcriptional activator(Gli3-190) and a processed form that acts as a transcrip-tional repressor (Gli3-83). Processing of Gli3 from the full-length activator form to the truncated repressor form is regu-lated by Hh signaling. Using western blotting and densito-metry to quantify protein levels in tissue extracted frommutant and wild-type embryos, we found that there appearedto be a direct relationship between Ift172 dosage and Gli3processing (Fig. 4C). In wild-type embryos, the ratio of pro-cessed to unprocessed Gli3 was 1.33. The ratios of pro-cessed to unprocessed Gli3 in avc1 mutants, avc1/wimcompound heterozygote mutants and wim mutants were0.39, 0.17 and 0.01, respectively (Fig. 4). This representeda 70.7% reduction of Gli3 processing in Ift172avc1 mutantembryos, an 87.2% reduction in Ift172avc1/wim embryos anda 99.2% reduction in Ift172wim embryos. Thus, theIft172avc1 allele disrupted the Hh signaling, but to a lesserextent than the Ift172wim allele. These results supportedthe conclusion that Ift172avc1 is a hypomorphic allele ofIft172 and causes significant but incomplete loss of embryo-nic Hh signaling.

The intersection between the requirement for cilia and Hhsignaling in heart development had not been demonstrated.We asked whether a decrement in Hh signaling couldaccount for the atrioventricular septal defects observed in

Figure 4. Hh signaling defects in avc1 mutants. (A and B) Whole-mountanalysis of Hh-signaling-responsive genes Ptch1 and Gi1 in E10 embryos.Ptch-lacZ expression (upper panels) recapitulated known Hh-signalingpattern in wild-type (A) embryos, and with qualitatively reduced level ofexpression in avc1 mutant littermates (B). Gli1 expression is demonstratedin a similar pattern by in situ hybridization and was qualitatively reduced inavc1 mutant embryos (middle and lower panels). The arrow denotes Gli1expression in the second heart field, behind the heart. (C) Full-length(Gli3–190) and processed (Gli3–83) forms of Gli3 were detected bywestern blot in wild-type, Ift172avc1, Ift172avc1/wim and Ift172wim embryos.The average processed Gli3–83 to full-length Gli3-190 protein ratios were1.33, 0.39, 0.17 and 0.01, respectively. (D and E) Hh-receiving atrialseptum progenitors were marked in R26RGli1CreERT2 embryos by the adminis-tration of tamoxifen at E7.5 and 8.5 and visualized at E13.0. In wild-typeembryos, the Hh-receiving lineage filled the atrial septum (D). In avc1mutant embryos, the Hh-receiving lineage was absent and a large atrioventri-cular septal defect was observed (E).



avc1 mutant embryos. Hh-receiving atrial septum progenitorcells were marked by Gli1 expression in Gli1:CreERT2;R26Rembryos (33) and their contribution to the atrial septum wasevaluated in wild-type and avc1 mutant embryos usinggenetic inducible fate mapping. Hh-receiving atrial septumprogenitors were marked in R26RGli1-CreERT2 embryos by theadministration of tamoxifen at E7.5 and E8.5 (33). TheHh-receiving lineage generated the atrial septum by E13.0 incontrol embryos (Fig. 4D). In contrast, the Hh-dependentatrial septum lineage was absent from avc1 mutant hearts atE13.0 (Fig. 4E). Thus, Ift172avc1 caused a structural cardiovas-cular phenotype, an atrioventricular septal defect, via deficientHh signaling.

Ift172 mutant embryos display truncated cilia

To evaluate the effects of reduced Ift172 expression on ciliastructure in vivo, we used scanning electron microscopy toanalyze the ventral node of Ift172avc1 mutant, Ift172avc1/wim

mutant and wild-type littermate embryos at E7.5 (Fig. 5).We observed a dose-dependent requirement for Ift172 in theproduction of full-length nodal cilia. The mean nodal cilialength was 3.56+ 0.05 mm in wild-type E7.5 embryos(Fig. 5A, D, G and H), 2.26+ 0.08 mm in Ift172avc1

embryos (Fig. 5B, E, G and H) and 0.81+ 0.02 mm inIft172avc1/wim embryos (Fig. 5C and F–H). Reduced Ift172dosage therefore impairs ciliogenesis in vivo at the ventralnode.

To better characterize the mechanistic basis for the quanti-tative decrement in Hh signaling observed in Ift172avc1

mutants, we evaluated the primary cilia of mutant mouseembryonic fibroblasts (MEFs). We predicted that fibroblastprimary cilia would be abnormal in the avc1 mutant settingand tested this hypothesis by isolating and comparing fibro-blasts from wild-type, Ift172avc1 and Ift172avc1/wim embryos.Analysis of the Ift172 allelic series revealed a dose-dependentrequirement for Ift172 in the synthesis of primary cilia. Usingacetylated tubulin immunohistochemistry, cilia were identifiedon 90.8% of wild-type cells (Fig. 6A, D and G), 41.5% ofIft172avc1 mutant cells (Fig. 6B, E and G) and 28.4% ofIft172avc1/wim cells (Fig. 6C, F and G).

The documented deficiency in Hh signaling appeared greaterin avc1 mutants than could be explained by the decreased per-centage of ciliated cells (avc1 mutants showed an �70%reduction in Gli3 processing, but only an �54% reduction inthe number of ciliated fibroblasts). We hypothesized that thecilia present on mutant cells may be abnormal and unable tosupport normal Hh signaling. We proceeded to structurallyand functionally evaluate the cilia present on avc1 and avc1/wim mutant MEFs. Quantitative analysis of primary cilialength revealed that there was a direct relationship betweenIft172 dosage and cilia length. Cilia on fibroblasts derivedfrom wild-type embryos were significantly longer than thecilia on cells derived from Ift172avc1 embryos which were inturn significantly longer than cilia on cells from Ift172avc1/wim

compound heterozygote embryos (Fig. 6H).Closer analysis of cilia length suggested that there were two

distinct populations of cilia present, full length and truncatedto approximately half length, rather than a normal distributionof cilia length within each genotype. Histogram analysis of thelength of cilia on Ift172avc1/avc1 fibroblasts demonstrated twopeaks, rather than a normal distribution of lengths. Ift172avc1

cilia were approximately equally divided between two popu-lations, a ‘full length’ population, with a peak centered at�2.5 mm, and a ‘truncated’ population, with a peak centeredat �1.5 mm (Fig. 6I). Analysis of wild-type fibroblast cilialength revealed a greater population of ‘full length’ cilia,with a much smaller population of ‘truncated’ cilia, althoughanalysis of Ift172avc1/wim fibroblasts revealed a large ‘trun-cated’ population, with a much smaller population of the‘full length’ cilia (Fig. 6I). Cilia of both types were presentin all genotypes, and the range of cilia length was nearly iden-tical between the three genotypes (Fig. 6I). This observationsuggested that the observed difference in mean cilia length

Figure 5. Truncated nodal cilia in Ift172 mutant mutants. (A–H) Scanningelectron microscopy was performed on the ventral node of E7.5 wild-type(A and D), Ift172avc1 (B and E) and Ift172avc1/wim (C and F) mutant littermateembryos. In contrast to the length of wild-type nodal cilia (3.56+0.05 mm),Ift172avc1/avc1 nodal cilia were foreshortened (2.26+0.08 mm, P , 0.001),and Ift172avc1/wim nodal cilia were severely truncated (0.81+0.02 mm, P ,

0.0001) (G and H). Four wild-type, four avc1/avc1 and five avc1/wimembryos were analyzed.



was due to varying the relative proportions between thenormal and truncated cilium populations, as opposed to a gen-eralized reduction in cilium length in mutant fibroblasts.

Ift172 mutant cilia demonstrate abnormal IFT88 and Gli2localization

We predicted that Ift172 mutant cilia might have impairedintraflagellar transport, resulting in aberrant cilia structureand Hh signaling. To test this hypothesis, we performedimmunocytochemistry with an antibody against IFT88, anessential component of IFT complex B, on wild-type,Ift172avc1 and Ift172avc1/wim cultured fibroblasts. It had beenpreviously reported that IFT88 concentrates at two siteswithin the cilium, one at the cilium base and one at thedistal tip (25). Interestingly, full-length cilia (.2 mm inlength) demonstrated normal IFT88 distribution, with foci ofstaining at the distal and proximal ends of the axoneme,regardless of genotype (Fig. 7). We found that 92.4% offull-length wild-type cilia (Fig. 7A–D and M), 85.7% of full-length Ift172avc1 cilia (Fig. 7E–H and M) and 83.3% of full-length Ift172avc1/wim cilia (Fig. 7I–M) showed normal IFT88distribution and there was no significant difference betweenthe distribution of IFT88 in full-length cilia of all genotypes(wild-type versus Ift172avc1, P ¼ 0.13; wild-type versusIft172avc1/wim, P ¼ 0.074). In contrast, the majority of trun-cated cilia showed only a single focus of IFT88 staining:only 42.1% of truncated Ift172avc1 cilia and 20.0% of trun-cated Ift172avc1/wim cilia showed the normal IFT88 distribution(wild-type versus avc1: P ¼ 5.54 × 10211 and wild-typeversus avc1/wim: P ¼ 1.53 × 10225, respectively). Even trun-cated cilia on wild-type MEFs showed the normal distributionof IFT88 only 64.7% of the time, significantly less often thanfull-length wild-type cilia (P ¼ 6.26 × 1026). These resultsidentified two distinct populations of primary cilia in culturedMEFs, full length and truncated, and documented abnormalIFT88 localization in the truncated population. A reductionin Ift172 dosage therefore established a truncated class ofcilia with abnormal IFT.

We hypothesized that the observed IFT defect in Ift172mutant cilia may impair the localization of Hh signaling com-ponents to the distal ciliary axoneme, providing a possiblemolecular explanation for the observed Hh signaling defects.We directly evaluated endogenous Gli2 localization by immu-nohistochemistry in wild-type and avc1 mutant MEFs to testthis hypothesis. Gli2 undergoes localization to the distal tipof the ciliary axoneme by an Hh-dependent mechanism(34,35). We found that after the activation of Hh signalingwith purmorphamine (PURM) (36), .60% of wild-typecontrol MEFs contained concentrated Gli2 at the distal tip ofthe ciliary axoneme (Fig. 8A–D and I–L). There was nodifference in the percentage of Gli2-positive wild-type full-length cilia (64+ 3.5%) versus truncated cilia (65+ 5.2%;P ¼ 1). However, Gli2 localized to significantly fewer ciliain Ift172avc1 mutant MEFs after PURM treatment (Fig. 8Q).Compared with wild-type cilia, Gli2 localized to significantly

Figure 6. Abnormal number and morphology of primary cilia in Ift172 mutantfibroblasts. (A–G) Fibroblasts were isolated from Ift172avc1 and Ift172avc1/wim

mutant and wild-type littermate embryos. After serum-starving to promotecilia growth, primary cilia were visualized in the cultured cells with acetylatedtubulin (red). Arrows identify primary cilia. Nuclei were stained with DAPI(blue). Nearly all wild-type fibroblasts were ciliated (90.8%) (A, D and G),although significantly fewer Ift172avc1 (41.5%) (B, E and G) and Ift172avc1/wim

(28.4%) (C, F and G) mutant fibroblasts were ciliated (P-values 8.12 × 102173

and 4.06 ×102239, respectively). (H) Ift172avc1 and Ift172avc1/wim mutant ciliawere significantly shorter on average than wild-type cilia (2.18 and 1.62versus 2.62 mm, P-values of 1.20 × 1026 and 1.71 × 10230, respectively). (I)Histogram analysis of primary cilia length demonstrates two distinct cilia popu-lations, a full-length population and a truncated population that are approxi-mately half normal length. Wild-type cilia were mostly full-length, Ift172avc1

cilia were divided evenly between the two populations and Ift172avc1/wim ciliaappeared mostly truncated.



fewer full-length mutant cilia (41+ 3.4%; P ¼ 0.003) andtruncated mutant cilia (28.5+ 2.2%; P ¼ 0.001) (Fig. 8E–H and M–Q). These results indicate that avc1 impairsHh-dependent Gli2 localization to the distal ciliary axoneme,a possible mechanism for the Hh signaling decrementobserved in avc1 mutant embryos.

DISCUSSION

We report that VACTERL-H may be added to the list of con-firmed ciliopathies in mice, demonstrating that avc1, a novel

hypomorphic allele of Ift172, results in VACTERL-H. Wedocument abnormalities in cilia structure, intraflagellar trans-port and Hh signaling that provide mechanistic insight intohow avc1 causes the VACTERL-H phenotype. Our resultsprovided empirical evidence for the previously suspected butunsubstantiated link between cilia, Hh signaling andVACTERL-H. We demonstrated for the first time the cellularmechanism causing a congenital heart defect in a cilia mutant:loss of an Hh-dependent lineage that generates the atrialseptum, resulting in AVSDs with common atrium. Further-more, by documenting the presence of a specific class of

Figure 7. Abnormal Ift88 localization in Ift172 mutant primary cilia. (A–M) Immunofluorescence was performed on serum-starved cultured fibroblasts fromwild-type (A–D), Ift172avc1 (E–H) and Ift172avc1/wim (I–L) embryos. Cilia were stained with acetylated tubulin (red) and IFT88 was stained with a specificanti-IFT88 antibody (green). Yellow arrows identify foci of Ift88 fluorescence. Size standard equals 3 mm. Cilia of each genotype were analyzed in twogroups, full length (.2 mm) and truncated (,2 mm). The majority of full-length cilia of all genotypes displayed the normal distribution of IFT88, with onefocus at the distal tip and one focus at the proximal tip (92.4% of wild-type cilia, 85.7% of Ift172avc1 cilia and 83.3% of Ift172avc1/wim cilia) (M). There wasnot a significant difference in IFT88 distribution between any of these groups (wild-type versus Ift172avc1 P ¼ 0.13; wild-type versus Ift172avc1/wim

P ¼ 0.074; Ift172avc1 versus Ift172avc1/wim P ¼ 0.49) (M). Truncated mutant cilia revealed a different distribution (M); the majority of Ift172avc1 truncatedcilia (57.9%) and the majority of Ift172avc1/wim truncated cilia (80.0%) had a single focus of IFT88 staining. IFT88 distribution was significantly differentbetween truncated cilia and full-length cilia of all genotypes. IFT88 distribution was also significantly different between truncated wild-type cilia and eithertruncated Ift172avc1 cilia (P ¼ 0.028) or truncated Ift172avc1/wim cilia (P ¼ 1.65 × 1026).



truncated cilia with disrupted IFT and Hh signaling in avc1mutants, we add a novel piece to the complex understandingof how cilia mutations impact the Hh signal transductionpathway and L–R determination.

The complete range of the VACTERL-H phenotypes isobserved in Ift172avc1 mutant embryos with variable pene-trance. Although several null alleles of IFT protein genesresult in the absence of cilia, hypomorphic ciliary mutationsallowing functional analysis of mutant cilia are less welldescribed. One previously described hypomorphic allele ofan IFT complex B protein, the ORPK allele of IFT88(reviewed in 30), allows survival to adulthood and a mildersubset of phenotypes than those caused by avc1. Like avc1mutants, ORPK mice have also been shown to have truncatedcilia. The ‘gasping’ (gsp) phenotype reported by Ermakov

et al. (37) demonstrates some similarities to VACTERL andto avc1, with abnormalities of foregut and hindgut, as wellas polydactyly. The gsp1, gsp3 and gsp6 lines were allreported to have truncated nodal cilia, although the responsiblegene has not been identified. Identification of avc1 as aloss-of-function mutation in an IFT complex B gene providesthe first identification of a monogenic cause for VACTERL-Hassociation, which may now be considered a syndrome.

The avc1 allele of Ift172 decouples the role of the cilium inL–R patterning from its role in Hh signaling: L–R axis determi-nation was preserved, whereas Hh signaling was significantlydisrupted. The generation of L–R asymmetry is at least partiallydependent on nodal flow established by rotating nodal monoci-lia (26), and L–R patterning abnormalities are common in ciliamutants (see reference 38 for partial list). For example,

Figure 8. Abnormal Gli2 localization in truncated avc1 mutant cilia. (A–P) Immunofluorescence was performed on serum-starved fibroblasts isolated fromIft172avc1 mutant embryos and their wild-type littermates. Fibroblasts were incubated with PURM to activate the Hh signaling pathway. Cilia were thenstained with acetylated tubulin (green) and Gli2 with a specific anti-Gli2 antibody (red). Full-length and truncated cilia were analyzed separately. (Q) Approxi-mately 64% of full-length or truncated wild-type cilia demonstrated Gli2 localization to the distal tip of the axoneme, whereas only 41% of full-length avc1mutant cilia (∗P ¼ 0.003) and 28% of truncated avc1 mutant cilia (∗∗P ¼ 0.001) demonstrated similar localization.



Ift172wim mutants demonstrated absent nodal cilia and random-ized L–R patterning [e.g. randomized heart looping (22)]. Incontrast, Ift172avc1 mutants demonstrated foreshortened nodalcilia but normal L–R patterning (e.g. normal heart looping inall cases). Our observations suggested that the proximal endof the cilium, maintained in avc1 mutants, may be sufficientto support normal rotation of nodal cilia and establishment ofnodal flow, although insufficient to support normal Hh signaltransduction. Although further studies are required to evaluatethis hypothesis more definitively, this model could explain thedecoupling of L–R axis formation from Hh signaling defectsobserved in avc1 mutant mice.

Our results suggest that decreased Ift172 dosage affects ciliasynthesis in two distinct ways: (i) cells are less likely toproduce cilia at all in the setting of decreased Ift172 dosageand (ii) the cilia that are formed are more likely to be truncatedwith abnormalities in IFT and Hh signaling. Fibroblasts withno Ift172 make no cilia, and carry out no Hh signaling trans-duction (22). Our analysis of the Ift172 allelic series utilizingour avc1 allele with the wim null allele of Ift172 suggests adose-dependent requirement of Ift172 for the synthesis offunctional primary cilia. Furthermore, we report that a hypo-morphic allele of Ift172 leads to disrupted IFT in the settingof a specific class of foreshortened cilia. In avc1 and avc1/wim mutant MEFs, we observed a distinct class of cilia thatare approximately half normal length (Fig. 6) with associatedmolecular abnormalities of the distal axoneme, e.g. loss ofIFT88 and Gli2 localization (Figs 7 and 8, respectively).Thus, as the Ift172 dosage was decreased, both the numberof ciliated fibroblasts and the percentage of full-length, fullyfunctional cilia decreased. The relative proportions of cellswith full-length, fully functional cilia, cells with truncated,dysfunctional cilia and cells with absent cilia will likely deter-mine the degree to which the Hh signal transduction pathwayis disrupted by any given cilia mutation.

These observations are consistent with a model of the mam-malian cilia as a composite structure composed of distinctfunctional units, with the distal tip specifically required forcilium-based signaling. Work in Caenorhabditis elegans hasdemonstrated that differences in the IFT molecular com-ponents determine the distinction between axonemal coreand distal tip, and mutants lacking the distal singlet tipsdemonstrate signaling defects (reviewed in 39). A similarmodel governing mammalian cilia-based signaling wouldpredict that cells with abnormalities of the distal ciliaryaxoneme would demonstrate defective signal transduction, aswe observed in Ift172 mutant MEFs. The observed quantitat-ive loss of Hh signaling and defective or absent distalaxoneme in Ift172 mutant cilia support a model requiringthe distal ciliary axoneme for Hh signal transduction. Aspecific defect of the distal axoneme of Ift172 mutant ciliain mice is consistent with the required role of IFT172 in thetransition of intraflagellar transport at the cilia tip of Chlamy-domonas (40). However, this model does not explain ouradditional observation that Gli2 localization is impaired innormal-length avc1 mutant cilia (Fig. 8Q). This observationimplies that IFT172 may also have a role in Gli2 traffickingand Hh signaling independent of its role in ciliogenesis.More work will need to be done to further elucidate the role

of IFT172 on the trafficking of Hh signaling moleculeswithin the ciliary axoneme.

The list of confirmed ciliopathies includes Bardet–Biedl,nephronophthisis, Senior–Loken syndrome, Alstrom syn-drome, Meckel syndrome, Joubert syndrome, oral–facial–digital syndrome type I, Jeune asphyxiating thoracic dystro-phy, Ellis–van Creveld syndrome, Leber congenital amauro-sis, Kartagener syndrome and polycystic kidney disease[reviewed in (41)]. Here we provide evidence that avc1, ahypomorphic mutation of Ift172, caused a ciliopathy withVACTERL-H-like features in mice. By providing a directlink between a single IFT gene mutation and VACTERL-Hphenotypes, this work illustrates the diversity of phenotypesthat can result from IFT gene mutations. IFT gene mutationshave been significantly under-represented in the human cilio-pathy literature. Establishing a Mendelian etiology forVACTERL-H in mice, we suggest that IFT genes, particularlyIFT complex B genes including Ift172, be considered candi-date genes for human VACTERL-H.

MATERIALS AND METHODS

Mouse strains

Avc1 was identified in a screen for recessive ENU-inducedmutations that caused perinatal lethality and structural heartdefects. Identification and characterization of wim has beenpreviously described (22). The Gli1CreERT2 line was obtainedfrom the Joyner Laboratory (Sloan-Kettering Institute, NY,USA). Activation of CreERT2 was accomplished by oralgavage with 2 mg of tamoxifen per dose in corn oil to pregnantdams, demonstrated to achieve optimal activation of CreERT2

without toxicity (33).Patched1-LacZ (B6;129-Ptch1tm1Mps/J) and R26R

(B6;129S4-Gt(ROSA)26Sortm1Sor/J) mice were obtained fromThe Jackson Laboratory, and genotyping was performed asdescribed (www.jax.org). All mouse experiments were per-formed in a mixed B6/129/SvEv background. All experimentsinvolving mice were carried out according to a protocolreviewed and approved by the Institutional Animal Care andUse Committee of The University of Chicago, in compliancewith the USA Public Health Service Policy on Humane Careand Use of Laboratory Animals.

Genetic mapping and molecular identification of avc1

SNPs that were previously identified as polymorphic betweeninbred strains of mouse, including C57BL/6J, FVB/NJ andC3H/FeJ (42) were genotyped on the Illumina platform (42)using DNA from affected mice. Additional polymorphicmarkers from publically available databases were utilized forhigh-resolution mapping. Avc1 was mapped to a 0.9 Mb inter-val on chromosome 5 between D5Mit229 and D5Mit420 in amapping cross with more than 1000 opportunities for recombi-nation. The only sequence alteration detected in the avc1-containing molecular interval was the A-to-G transition at pos-ition +3 in the Ift172 exon 24 splice donor site.



Embryo dissection and X-Gal staining

Embryos were dissected from maternal tissue samples and tailsamples were taken for genotyping. Mice or dissected organswere then fixed for 1 h in 4% paraformaldehyde and stainedwith X-gal staining solution [5 mM K3Fe(CN)6, 5 mM

K4Fe(CN)6, 2 mM MgCl2, 0.02% NP-40, 0.01% deoxycholate,0.1% X-Gal in PBS] when appropriate or fixed in 10% neutralbuffered formalin if no b-galactosidase-producing allele waspresent. To facilitate staining, embryos older than E10.5were dissected open prior to staining. Mice were embeddedin paraffin and sectioned at 5 mm. If X-gal stain waspresent, slides were counter-stained with 50% eosin for 1 s.If no X-gal stain was present, the slides were stained withhematoxylin and eosin.

Antibody production

Chemically synthesized peptides corresponding to KLRRDYYQWLMDTQQEER were sent to Pocono Rabbit Farm andLaboratory, Inc., for IFT172 antibody production in rabbitsand antibody purification. The synthesized peptide correspondsto amino acids 738–755 of IFT172 encoded in exon 22 (NCBIm37 mouse assembly) and shared by both wild-type and avc1proteins.

Western blotting

Western blots were performed in triplicate. Mouse embryoswere harvested in PBS at 48C, an aliquot taken for genotypingand the remainder transferred into 0.5 ml of RIPA lysis buffermaintained at 48C (Roche CompleteTM protease inhibitor,0.5 M Tris–HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid,10% NP-40, 10 mM EDTA). Tissues were lysed with rotor–stator-type homogenizer for 30 s. Lysates were rotated for15 min and centrifuged for 15 min at 12 000g. The resultingsupernatants were removed, assayed for protein concentration,normalized by the addition of RIPA buffer and diluted intoLaemmli sample buffer (Bio-Rad, 161-0737EDU). Theresulting samples were run into 8% Tris–glycine gels forSDS–PAGE and transferred to nitrocellulose blots for immuno-detection. Following block (5% milk/TBST for 30 min) andwash (TBST 3×), primary antibodies (a gift of B. Wang)were added at 1:100 in 2.5% BSA/TBST and incubated over-night at 48C. Alkaline phosphatase-conjugated secondary anti-body was utilized and blots were detected with an AmershamECL-Plus detection kit (RPN2124).

Bone staining

Bone and cartilage staining was performed on E18.5 embryosas described by McLeod (43).

Primary MEFs

MEFs were isolated from Ift172avc1/avc1 embryos at E13.5 andIft172avc1/wim embryos at E12.5. Culture conditions were asdescribed by Ocbina and Anderson (28).

Immunofluorescence

Cells were grown to confluence on eight well-chamberedslides (Lab-Tek II). Fixation and immunofluorescence stainingwere performed as described by Ocbina and Anderson (28),with the following minor modifications: blocking solutionwas composed of PBS with 10% v/v normal donkey serumand 0.1% v/v Triton X-100. All antibodies were diluted in1:1 blocking solution and PBS. The following primary anti-bodies were used: mouse anti-acetylated-alpha-tubulin(Sigma-Aldrich, T7451) at a dilution of 1:1000; rabbitanti-Ift88 (a gift from B. Yoder, University of Alabama, Bir-mingham, AL, USA) at a dilution of 1:500 and rabbitanti-Gli2 (a gift from Paotien Chuang) at a dilution of1:400. Alexa488, Alexa568, goat anti-mouse AlexaFluor 488and goat anti-rabbit AlexaFluor 594-conjugated secondaryantibodies (Invitrogen) were each used at a dilution of 1:500.

Electron microscopy

E8.0 embryos were dissected in PBS at room temperature andimmediately fixed in 2.5% glutaraldehyde and 2% PFA in0.1 M sodium cacodylate buffer, pH 7.4 (Electron MicroscopySciences), for at least 1 h at room temperature. Embryos werestored in a fixative for an additional 24 h at 48C before proces-sing as described in Huangfu and Anderson (44). Scanningelectron micrograph images were taken on a Zeiss SUPRA25 FESEM.

In situ hybridization

In situ hybridization was performed using digoxigenin-labeledprobes. Protocol was as described in (45), with the followingchanges: embryos were washed six times in maelic acidbuffer for 1 h (MAB; 0.1 M maelic acid, pH 7.5, 0.15 M

NaCl, 0.1% Tween-20 and 0.002 M levamisole), followed bya 16 h overnight wash at room temperature. Color reactionswere allowed to develop overnight, and images were takenprior to storage in 80% glycerol. The in situ probe for Ift172message spanned the 30th to 40th exons and for Gli1 asdescribed (33).

ACKNOWLEDGEMENTS

The authors thank Bradley Yoder and the University ofAlabama at Birmingham Hepato/Renal Fibrocystic DiseasesCore Center (UAB HRFDCC DK074038) for the IFT88 anti-body.

Conflict of Interest statement. None declared.

FUNDING

This work was funded by grants from the NIH [R01HL092153 (I.P.M.)], the March of Dimes (I.P.M.), the AHA(I.P.M.) and the Schweppe Foundation (I.P.M.).



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a novel murine allele of intraflagellar transport protein 172 causes

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