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Am J Pathol. Apr 2006; 168(4): 1288–1298.
PMCID: PMC1606550

ADP-Ribosylation Factor-Like 3 Is Involved in Kidney and Photoreceptor Development

Abstract

ADP-ribosylation factor-like 3 (Arl3) is a member of a small subfamily of G-proteins involved in membrane-associated vesicular and intracellular trafficking processes. Genetic studies in Leishmania have shown that the Arl3 homolog is essential for flagellum biogenesis. Mutations in a related human family member, Arl6, result in Bardet-Biedl syndrome in humans, which is characterized by genital, renal, and retinal abnormalities, obesity, and learning deficits. As part of our large-scale phenotypic screen, mice deficient for the Arl3 gene were generated and analyzed. Arl3 (−/−) mice were born at a sub-Mendelian ratio, were small and sickly, and had markedly swollen abdomens. These mutants failed to thrive, and all died by 3 weeks of age. The (−/−) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, which is characteristic of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease. Absence of Arl3 was associated with abnormal epithelial cell proliferation and cyst formation. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration as early as postnatal day 14. These results are the first to implicate Arl3 in a ciliary disease affecting the kidney, biliary tract, pancreas, and retina.

Polycystic kidney disease (PKD) is a relatively common inherited disease and is one of the leading causes of end-stage renal disease in humans. PKD is characterized by the presence of cysts in the kidney, liver (50%), and pancreas (10%). Two forms of PKD are recognized: autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). ADPKD occurs in 1 in 1000 live births, is slowly progressive, and most often leads to renal failure in adulthood. In contrast, ARPKD is rare, with an estimated incidence of 0.5 to 1 in 20,000 live births,1 and progresses rapidly to renal failure in infants and children.2 ARPKD is also frequently associated with biliary dysgenesis, which is characterized by dilatation of intrahepatic bile ducts and portal fibrosis.

Several mouse models develop lesions bearing a close resemblance to human PKD in terms of pathogenesis and disease progression.3–7 These models have proven very useful in elucidating the roles of altered cell proliferation, cell differentiation, extracellular matrix composition, ionic transport, and oncogene expression in the development of PKD. The best characterized models are the congenital PKD mouse (cpk)3 and the Oak Ridge PKD mouse (Tg737/orpk).4 Although murine models of PKD share many features in common in the kidney, there is some variation in extrarenal cystic disease. Mandell and colleagues5 described a congenital polycystic kidney mutation (cpk) in the mouse that closely resembles human ARPKD because of concurrent cystogenesis in the liver and pancreas. These mutants die from a rapid progressive renal insufficiency and do not develop biliary ductal plate malformations.6,7 PKD associated with a hypomorphic allele of the Tg737 polaris gene also closely resembles the clinical features of human ARPKD. The (Tg737/orpk) mutants develop renal cysts, biliary-dysgenesis, pancreatic cysts, and skeletal defects.8 In addition to these defects, the targeted deletion of the Tg737 gene causes loss of embryonic nodal cilia, disruption of LR axis specification, and embryonic lethality.8

Many of the proteins involved in human PKDs have been localized to the primary cilia, including PKD1, PKD2,9,10 fibrocystin,11 and inversin.12 Similarly, PKD-related proteins in mice (polycystin-1 and -2, cystin, and polaris) have also been localized to the primary cilia.9,13 These findings suggest that the primary cilium plays a key role in normal physiological functions of renal epithelia and that defects in ciliary function contribute to the pathogenesis of PKD. In particular, in the renal tubule lumen, the projecting primary cilia are believed to act in a sensory role in cilia function.14 More recently others have suggested that the intraflagellar transport protein, IFT88, and the Caenorhabditis elegans homolog of Tg737 are essential for photoreceptor assembly and maintenance.14–16 Here the localized protein expression in cilia coupled with the process of intraflagellar transport (IFT), which involves the transport of cargo from the axoneme to the flagellar or cilia tip, is associated with Tg737/polaris protein function. Proper assembly and maintenance of cilia or flagella are dependent on IFT.17,18

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins are small GTP-binding proteins that regulate a wide variety of intracellular signaling and vesicular trafficking pathways19 that may include the movement of cellular components within cilia.20 Relatively little has been reported about the in vivo function or the physiological role of Arl3. Arl3 is a Ras-related small GTP-binding protein, is similar to other ADP-ribosylation factor (ARF) family members, binds guanine nucleotides, but lacks ARF activity.21 Interestingly, an Arl3 homolog has been localized within the Leishmania flagella and is essential for flagellum biogenesis.22 In addition, Arl3 is found in the ciliary compartment groups implicated in outer segment development and ciliogenesis23 as well as in humans in the connecting cilium of retinal photoreceptor cells.24 Arl3 has been shown to interact with the X-linked retinitis pigmentosa protein (RP2), and mutations in RP2 result in a severe form of retinal degeneration involving the rod photoreceptors.25 This association suggests the possibility that Arl3 dysfunction might also be involved in the pathobiology of retinal degenerative diseases.

Using embryonic stem (ES) cells derived from the OmniBank gene-trap library,26 we have discovered a PKD defect and abnormal photoreceptor development in mice lacking Arl3. These results provide the first genetic evidence that Arl3 is associated with PKD and implicate Arl3 in photoreceptor development. We describe these pleiotropic defects, which implicate Arl3 as another genetic model that supports a growing ciliary-related disease paradigm.

Materials and Methods

Generation of Arl3 Mutant ES Cells and Mice

The construction of the OmniBank gene-trap library has been described.26,27 Arl3 mutant mice were generated by microinjection of OST 263303 ES cells into host blastocysts using standard methods.28 The precise genomic insertion site of the retroviral gene-trapping vector in the Arl3 gene was determined by inverse genomic polymerase chain reaction (PCR).29

Mouse Genotyping

Oligonucleotide primers (LTR2, 5′-AAATGGCGTTACTTAAGCTAGCTTGC-3′; A 5′-GGTCATCTGGCCCATAACTAATCA-3′; and B 5′-CCGGGACTGGAGTTAGTAGTGGTT-3′) were used in a multiplex reaction to amplify corresponding Arl3 alleles (wild-type, +/+; mutant, −/−). Diluted mouse tail lysate containing ~20 ng of genomic DNA was used as a template for PCR in a 50-μl reaction volume containing 3.5 mmol/L MgCl2. PCR conditions were as follows: 94°C for 15 seconds, 65°C for 30 seconds (then decrease 1°C per cycle), 72°C for 40 seconds, for 10 cycles; followed by 94°C for 15 seconds, 55°C for 30 seconds, 72°C for 40 seconds, for 30 cycles. Amplified products were separated on 3% agarose gels.

Arl3 Reverse Transcriptase (RT)-PCR

RNA was extracted from kidney and spleen using a bead homogenizer and RNAzol (Ambion, Austin, TX) according to the manufacturer’s instructions. Reverse transcription was performed with SuperScript II (Invitrogen, Carlsbad, CA) and random hexamer primers, according to the manufacturer’s instructions. PCR amplification (95°C, 30 seconds; 59°C, 45 seconds; 70°C, 60 seconds.) was performed for 30 cycles using primers complementary to exons 1 and 2 of the Arl3 gene, flanking the insertion site of the vector (primer C: 5′-GCGGAGG AGGAGGAGGAGGGACTCG-3′, primer D: 5′-GTAGTCTTGCCCGCG TTGTCCA-3′). Control primers to the mouse β-Actin gene (accession number M12481) were 5′-GGCTGGCCGGGACCTGACGGACTACCTCAT-3′ and 5′-GCCTAGA AGCACTTGC GGTGCACGATGGAG-3′. Arl3 RT-PCR products were verified by sequencing.

Mouse Husbandry

Mice were housed in a barrier facility at 24°C on a fixed 12-hour light/dark cycle and were fed rodent chow no. 5001 (Purina, St. Louis, MO) ad libitum. Procedures involving animals were conducted in conformity with Institutional Animal Care and Use Committee guidelines that are in compliance with state and federal laws and the standards outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

Tissue Pathology

Tissues were collected from knockout mice and age-matched control mice up to 3 weeks of age and were immersion-fixed in 10% neutral buffered formalin. All tissues were embedded in paraffin, sectioned at 4 μm, mounted on positively charged glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA) and stained with hematoxylin and eosin (H&E) for histopathological examination. Immunohistochemistry was also performed on formalin-fixed, paraffin-embedded tissues. We detected proliferating cells using antibodies to Ki-67 protein.

Ki-67 Immunohistochemistry

For immunohistochemistry, 4-μm sections were first deparaffinized in xylene and rehydrated in phosphate-buffered saline (PBS); then endogenous peroxidase activity was blocked by incubation in 3.0% hydrogen peroxide in PBS for 5 minutes. For antigen retrieval, sections were immersed in citrate buffer (pH 6.0) for 20 minutes in a steamer preheated to 95°C. Primary antibody (1:30 rat anti-mouse Ki-67; DAKO, Glostrup, Denmark) was applied for 1 hour. After rinsing, sections were incubated for 1 hour in a 1:400 dilution of the biotinylated secondary antibody (rabbit anti-rat IgG; Vector Laboratories, Burlingame, CA). Bound antibodies were detected by an avidin-biotin complex method (Elite ABC kit, Vector Laboratories) with 3,3′-diaminobenzidine as chromogenic substrate following the manufacturer’s instructions. Negative controls included substitution of nonimmune serum for the primary antibody and omission of the primary antibody. After counterstaining with hematoxylin, sections were mounted with Permount (Fisher Scientific).

Ophthalmic Histopathology

Ophthalmic histopathology was conducted on mice at different postnatal ages. Mice were euthanized, and eyes were removed and placed in Davidson’s fixative (Poly Scientific, Bayshore, NY) overnight at room temperature. The following morning, eyes were processed in paraffin, cut at a thickness of 5 μm, and stained with H&E. For immunohistochemistry, mice were deeply anesthetized with (per) ketamine (7.5 mg/ml), xylazine (0.38 mg/ml), and acepromazine (0.074 mg/ml), delivered at ~10 ml/kg body weight followed by perfusion with 4% paraformaldehyde in 0.1 mol/L sodium phosphate-buffered saline (PBS) (pH 7.2). Eyes were removed and incubated in PBS containing 25% sucrose and embedded in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC). Sections were cut at a thickness of 12 to 16 μm and mounted on Fisher Superfrost/Plus slides.

Fluorometric Terminal dUTP Nick-End Labeling (TUNEL), Anti-Rhodopsin, and PNA Assays

Dying cells were detected using the Dead-End fluorometric TUNEL system (catalog no. G3250; Promega, Madison, WI) per the manufacture’s recommendations. Primary antibodies were diluted in 5% normal goat serum (Vector Laboratories) in PBS. Dilutions were as follows: mouse anti-rhodopsin at 1:100,000 (Chemicon, Temecula, CA) and fluorescein-conjugated peanut agglutinin (PNA) at 1:250 (Vector Laboratories). Alexa 594 goat anti-mouse (diluted at 1:400) was used to detect primary mouse antibodies. Coverslips were applied using mounting media (Vectashield, Vector Laboratories). Digital images were obtained with a Hamamatsu ORCA II cooled charge-coupled device camera mounted on an Olympus BX60 microscope. Images were acquired in Adobe Photoshop version 6.0 and saved at a resolution of 300 ppi. Figures were assembled in Photoshop, and minimal adjustments were made to the figure contrast to obtain the best micrograph.

Electron Microscopy Studies

For the electron microscopy studies, mice at postnatal day 9 were anesthetized as described above and perfused with 3.5% glutaraldehyde in 0.1 mol/L phosphate buffer. Eyes were removed, and retinas were incubated in the same fixative overnight. Tissue was then incubated in 2% osmium tetroxide, stained with 2% uranyl acetate, and embedded in Spurr’s resin. Semithin sections were stained with 1% toluidine blue. Ultrathin sections were obtained and collected in formvar-coated slot grids and subsequently stained with lead citrate.

Results

Mouse ES Cells

ES cells carrying a mutation in the Arl3 gene (accession number NM_019718) were obtained from OmniBank, a library of gene-trapped ES cell clones identified by a corresponding OmniBank Sequence Tag (OST).26,27 Inverse genomic PCR29 analysis of DNA from this clone confirmed the insertion of the gene-trapping retroviral vector in intron 1 of the Arl3 gene on chromosome 19, downstream of the initiation codon in exon 1 (Figure 1A). OST 263303 cells were used to generate mice heterozygous for the Arl3 mutation using standard methods,28 and genotypes were confirmed by multiplex PCR on genomic DNA (Figure 1B). Interbreeding of Arl3 heterozygote mice gave rise to fewer (−/−) animals than predicted by a standard Mendelian distribution with 56 wild-type (+/+), 90 heterozygous (+/−), and 20 homozygous (−/−) mice. We confirmed the disruption of Arl3 transcription by the gene-trapping vector by reverse transcriptase (RT)-PCR using primers complementary to Arl3 exons 1 and 2, flanking the site of integration of the vector. Arl3 (+/+) transcript was not detected in tissues of Arl3 (−/−) mice (Figure 1C).

Figure 1
A: Gene trap mutation of the Arl3 gene. SA, splice acceptor sequence; Neo, neomycin resistance gene (arrow indicates transcription start site). B: Genotyping strategy. Primers A and B flank the genomic insertion site in intron 1 and amplify a product ...

Renal Pathogenesis

Male and female (−/−) Arl3 mice were identifiable within a few days of birth by a pronounced distention of their flanks caused by severe bilateral renomegaly (Figure 2, A and B). The Arl3 (+/−) mice were normal in all phenotypic screens between 8 to 16 weeks of age (data not shown). This analysis included observations in radiology, immunology, blood chemistry, behavior, and cardiology. Grossly, all Arl3 (−/−) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, characteristic of the renal-hepatic-pancreatic dysplasia reported in ARPKD. The renal cysts continued to enlarge after birth and the mice were euthanized before reaching 3 weeks of age (Figure 2, C and D). The kidneys were uniformly enlarged, light tan to white in color, and had a spongy consistency. Innumerable translucent and minute cystic structures were observed throughout the renal cortex and medulla. The gall bladder was severely distended, and extrahepatic bile ducts were dilated. In the liver the portal tracts were prominent. The pancreas was very small and consisted of dilated ducts and cysts surrounded by reduced amounts of parenchymal tissue. All other tissues examined displayed no pathologies and no laterality defects were seen in the (−/−) Arl3 mice.

Figure 2
A: A normal age-matched (+/+) mouse on the bottom shows the small size of Arl3 (−/−) on top with distended sides. B: A typical Arl3 (−/−) mouse at 3 weeks of age is shown, note the enlarged flanks. C: An ...

Microscopic examination showed that normal renal parenchyma was almost completely effaced by myriad fluid-filled cysts in both the cortex and medulla (Figure 3B). Renal cysts were identified in all segments of the nephron, including proximal and distal tubules, collecting ducts, and glomerular Bowman’s spaces. The collecting duct cysts became more prominent with increasing age, and by postnatal day 10 only a few scattered foci of small or atrophic tubules remained interspersed among the cysts. A single layer of low columnar, cuboidal, or squamous epithelial cells lined the distended cystic tubules. Some of the renal tubular cysts contained small amounts of eosinophilic material and cellular debris but most were empty. Most of the visible glomeruli were located at the edge of large cysts formed by dilation of Bowman’s capsule, but a few immature noncystic glomeruli were located within the superficial cortex (Figure 3B).

Figure 3
Representative photomicrographs of kidneys, pancreas, and liver from Arl3 (+/+) and (−/−) mice at postnatal day 10. A: In (+/+) mice, there are numerous Ki-67-positive cells in the densely packed cortical ...

Renal Immunohistochemical Detection of Ki-67 Antibody

Immunohistochemical detection of Ki-67, a nuclear nonhistone protein present only in proliferating cells, revealed significant differences between postnatal day 9 (−/−) Arl3 knockout mice and age-matched (+/+) controls in the kidneys, gall bladder, extrahepatic common bile ducts, and pancreatic ducts. As expected in rapidly growing mice of this age, proliferating cells (Ki-67-positive) were numerous in the renal cortex of both (+/+) and (−/−) mice. However, marked differences between (−/−) mice and (+/+) mice were noted in the collecting duct epithelium of the renal medulla and papilla. In (+/+) littermates, Ki-67-positive cells were relatively rare in the kidney medulla (Figure 3A). In contrast, in Arl3 knockout mice, numerous proliferating cells lined the cystic medullary collecting ducts as well as the cortical tubules and cystic glomeruli (Figure 3C).

Pancreatic Lesions and Immunohistochemical Detection of Ki-67 Antibody

Prominent lesions were also present in the pancreas of Arl3 knockout mice. There was a marked reduction in total pancreatic mass because of atrophy and hypoplasia of exocrine pancreas tissue. Distinct pancreatic lobules were absent, and most remaining pancreatic acini were disorganized and consisted of small irregular nests of cells surrounded by loose interstitial tissues (Figure 3D). There was widespread cystic dilatation of intralobular pancreatic ducts, and in some areas the duct walls were thickened by an inflammatory infiltrate and fibrosis. Immunohistochemistry showed that there were very few Ki-67-positive epithelial cells lining the normal pancreatic ducts of (+/+) mice (not shown). However, in some areas of the (−/−) mice, more than 80% of the cystic pancreatic duct epithelial cells were Ki-67-positive (Figure 3E). These results are consistent with an association between widespread cystic dilatation of pancreatic ducts and increased cell proliferation. Interestingly, even with the atrophy and loss of exocrine pancreas tissue, a few of the cells lining the cystic pancreatic ducts were also positive for insulin (Figure 3F).

Biliary Lesions and Immunohistochemical Detection of Ki-67 Antibody

In all (−/−) mice, gross and microscopic lesions consistent with ductal plate malformation were present in the biliary tract. The most notable lesion was the massive distention of the gall bladder and extrahepatic common bile ducts. Within the portal areas of the liver, there were increased numbers of intrahepatic bile ducts that were often distorted by multiple segmental and saccular dilatations (Figure 3H). In most areas, there was only a slight increase in periductular connective tissue. However, in some areas the dilated bile ducts contributed to stagnation of bile and ascending bacterial infections, resulting in a multifocal acute suppurative cholangitis. Portal inflammation and fibrosis were present in the areas where dilated intrahepatic bile ducts were filled with degenerating neutrophils and bacteria. In Arl3 knockout mice, Ki-67 immunohistochemistry labeled the majority of epithelial cells lining the gall bladder and some extrahepatic bile ducts but relatively few cells lining the intrahepatic ducts. In contrast, in (+/+) littermates (not shown), few cells lining the gall bladder or extrahepatic biliary ducts were Ki-67-positive. This again suggests that rapid cell proliferation promotes the massive distention of the gall bladder and extrahepatic common bile ducts. Overall, these results indicate that the pathological deterioration of Arl3 neonates is because of abnormal duct development in the kidney, liver, and pancreas.

Ophthalmological Histopathology

The general histological appearance of retinas obtained from (−/−) mice is similar to that in either (+/+) or (+/−) littermates at postnatal day 7 (not shown) or postnatal day 9 (Figure 4). The ganglion cell layer and inner nuclear layer (INL) are apparent, and the outer nuclear layer (ONL) is populated with photoreceptors in both the (+/+) (Figure 4A) and (−/−) mice (Figure 4B). At the light microscopic level, developing inner segments (ISs) and outer segments (OSs) of photoreceptors are apparent above the ONL in (+/+) mice (Figure 4A). In contrast, at postnatal day 9 the ISs and OSs appear rudimentary at this age in mice lacking Arl3 (Figure 4B) compared to those of (+/+) controls.

Figure 4
Histological and ultrastructural appearance of retinas obtained from postnatal day 9 mice. A: (+/+) Retina stained with H&E reveals three cellular layers (ONL, outer nuclear layer; INL, inner nuclear layer; and GCL, ganglion cell ...

Electron microscopy was performed to identify ultrastructural abnormalities in mice lacking Arl3 compared to their (+/+) littermates. The connecting cilium is a nonmotile structure that functions as a conduit for transport of proteins and lipids between the IS and OS in photoreceptors. Appearance of the connecting cilium occurs during the first postnatal week in mice, with subsequent formation of outer disks containing the photopigment rhodopsin.30 At postnatal day 9 in (+/+), ISs are packed with mitochondria, and they contain basal bodies (Figure 4C, arrow) associated with the connecting cilia (Figure 4C, arrowheads). Photoreceptor OSs (Figure 4C, asterisks) are observed in (+/+) mice at all retinal locations examined. Mice deficient in Arl3 developed ISs that contain mitochondria, basal bodies, and rudimentary connecting cilia (Figure 4D, arrows). However, Arl3-deficient mice exhibit a paucity of OSs at all retinal locations examined. Disks that were present appeared rudimentary compared to those disks in the postnatal day 9 (+/+). This finding suggests that Arl3 is involved in the development of OS disks.

At postnatal day 13 cone OSs can be identified with peanut agglutinin stain (PNA) conjugated to fluorescein. In (+/+) mice PNA reacted strongly with both the IS and OS associated with cone photoreceptors (Figure 5A). In contrast, (−/−) animals lacked any detectable cone OSs, although ISs were strongly labeled with PNA (Figure 5B). Rods were examined with an antibody recognizing rhodopsin. In (+/+) mice intense rhodopsin immunoreactivity was associated with rod OSs (Figure 5C). Weaker immunoreactivity was detected in IS at the dilutions of the antibody used in these studies. In contrast, intense rhodopsin immunoreactivity was associated with cell bodies of rod photoreceptors in mice lacking Arl3 (Figure 5D). Specific labeling of rod OSs was not observed in the region located adjacent to the retinal pigment epithelium. These results suggest that development of both rods and cones is impaired in mice lacking Arl3.

Figure 5
Analysis of cone and rod photoreceptors in retinas obtained from postnatal day 13 mice. Cones were visualized in the (+/+) (A) or (−/−) (B) retina with PNA conjugated to fluorescein. In (+/+) retina, the ...

Mutations that disrupt the development of photoreceptors or that impair interactions between photoreceptors and the retinal pigment epithelium are often associated with an increase in TUNEL-positive cells and death in the photoreceptor layer.31 To determine whether Arl3 deficiency is associated with an increase in photoreceptor death, TUNEL histochemistry was performed on postnatal day 13 (+/+) and (−/−) Arl3 mice. At this age in (+/+) mice, TUNEL-positive cells were located in the INL [Figure 6, A and B (arrows)], reflecting naturally occurring cell death during retinal development.32 Few TUNEL-positive cells were apparent in the ONL at this age in (+/+) mice. In contrast, absence of Arl3 resulted in a dramatic increase in TUNEL-positive cells in the ONL (Figure 6D, arrowheads). The relative number of TUNEL-positive cells in the INL remained comparable to that in (+/+) mice (Figure 6D, arrows). These results imply that Arl3 is critical for photoreceptor survival during the second postnatal week in mice.

Figure 6
TUNEL-histochemistry on sections obtained from postnatal day 13 mice. A: Nomarski image of the (+/+) retina shown in B. The optic nerve head is at the top left of the image. B: Several TUNEL-positive cells (arrowheads) are present in the ...

Discussion

Arl3 (−/−) mice exhibit a pleiotropic phenotype characterized by cysts in the kidney, liver, and pancreas and impaired photoreceptor development. Collectively these findings closely resemble those in humans and in mouse models associated with cilia dysfunction. For example, in the Tg737/orpk mutant mouse, the liver, kidney, pancreas, and eye defects resemble those found in the Arl3 (−/−) mutant mice.15 Both the Tg737/polaris and Arl3 genes are expressed in cilia, and our initial finding of PKD pathology in the Arl3 (−/−) mice reinforced this relationship. More generally, we suggest that the constellation of phenotypes we have observed in Arl3 (−/−) mice is likely to place Arl3 in the growing group of ciliary proteins associated with an overlapping set of phenotypes.33–35

Mutations that disrupt the assembly and/or physiological function of the primary apical cilium of tubular epithelia have been shown to be involved in the pathogenesis of PKD. Human proteins involved in PKD have been localized to the primary cilia, including PKD1, PKD2,9,10 fibrocystin,11 and inversin.12 Likewise, murine PKD-related proteins polycystin-1 and -2, cystin, and polaris have also been localized to the primary cilia.9,13 In fact, most of the identified human genes disrupted in PKD encode proteins that have been localized to the primary cilia of renal tubular epithelial cells.36,37 A role for specialized primary cilia has been proposed for the chemosensory, photosensory, and mechanosensory functions of olfactory neurons, retinal photoreceptors, and renal epithelium, respectively.33,38 We speculate that the mechanosensory renal primary cilia that respond to shear forces associated with luminal flow and thereby modulate renal tubular epithelial cell proliferation and differentiation are dysfunctional in the Arl3 mutant mice.

The primary epithelial cilium is important for differentiation of polarized epithelial cells, and both structural and functional defects in cilia result in PKD.13 The role of primary cilia in the pathogenesis of PKD was first suggested by findings in Tg737/orpk mice. Mutation of polaris (Tg737/orpk), which is localized in ciliary axonemes and basal bodies, interferes with the assembly of primary cilia, resulting in short stunted primary cilia in the kidney, retina, and at the embryonic node.16,34 The assembly, maintenance, and function of cilia depend on IFT particles or rafts containing protein cargo that are transported bidirectionally along the ciliary axoneme.39 Kinesin-II, a motor protein, moves anterograde to the tip of the cilium. The tissue-specific inactivation of the kinesin KIF3A subunit abolishes anterograde transport and results in viable offspring dying of PKD by 21 days.40 The morphologically normal cilia of cpk (cystin), inv (inversin), and Pkd2 mutant mice12,35,41,42 are co-localized to the primary cilia, and cystin expression overlaps with polaris.13,43 Moreover, fibrocystin co-localizes with polycystin-2 in the basal bodies and primary cilia of renal cells. Arl3’s status as a small GTP-binding protein suggests that it may regulate intracellular signaling, vesicular trafficking pathways,19 and/or movement of cellular components within or along cilia.20 We speculate that Arl3 may serve as a regulator in the process to polarize the ciliated epithelial cells and prevent mislocalization of critical proteins via IFT. Our observation of the mislocalization of rhodopsin in Arl3 (−/−) rod cell bodies supports this hypothesis.

Controlled proliferation and differentiation of epithelial cells is critical for the maintenance of normal renal tubule diameter and structure. It is believed that structural and/or functional defects in the mechanosensory primary apical cilia disrupt normal cell cycle regulation or differentiation of renal epithelial cells. Our findings of increased cell proliferation in the collecting ducts of the kidney and in the cystic pancreatic ducts, external hepatic ducts, and gall bladder (Figure 3, D and E) are consistent with the hypothesis that PKD is the result of disrupted cell-cycle regulation and differentiation of renal epithelial cells. Interestingly, primary cilia may have a sensory function that helps to regulate intracellular calcium concentration ([Ca2+]i) and maintain the differentiated renal epithelial phenotype.44 Maintenance of this phenotype is characterized by controlled fluid secretion and cell proliferation requiring precise functional coordination of cAMP and Ras/Raf/MEK/ERK signaling by [Ca2+]i. Primary cilia-localized proteins mutated in human ADPKD and ARPKD, as well as in several animal models, are thought to have a sensory function and contribute to the regulation of the ([Ca2+]i). Therefore, the mutations causing PKD may hinder the negative feedback mechanism controlling cAMP and Ras/Raf/MEK/ERK signaling and result in increased fluid secretion and cell proliferation. Arl3 is an Arf subfamily member of the Ras protein superfamily, so it will be interesting to study the potential for aberrant signaling by [Ca2+]i in the Arl3 (−/−) mouse.

The importance of abnormal proliferation and/or differentiation of renal epithelium in the pathogenesis of PKD has been noted in many systems. Cell responses to the primary cilia mechanoreceptors appear to help maintain the ratio of normal tubule-duct diameter to luminal flow by modulating epithelial cell proliferation and differentiation. With dysfunctional or absent primary cilia, the loss of sensory signals that normally inhibits cell proliferation may give rise to increased cell division and tubular dilatation. For example, renal cells lacking functional mouse KIF3A do not have primary cilia and exhibit increased proliferation and apoptosis, apical mislocalization of the epidermal growth factor receptor, increased expression of β-catenin and c-myc, and inhibition of p21(CIP1).40 In human ADPKD, elevated expression levels of the proto-oncogene c-myc expression are also seen.45 Rodent models of PKD using transgenic mice overexpressing c-myc in the renal tubular epithelium correlate with increased proliferation rates.45,46 Our findings in the Arl3 (−/−) mice showing that tubular dilatation is associated with proliferation of cells lining the collecting ducts (Figure 3C), suggests a role for Arl3 in modulating cell proliferation.

The association of renal and retinal diseases with ciliary dysfunction is becoming increasingly more appreciated.47 Several lines of circumstantial data suggest that Arl3 might be important for retinal photoreceptor biology. Arl3 is predominantly localized in the connecting cilia in both rod and cone photoreceptors and co-localizes with microtubules in vitro.24 Moreover, Arl3 interacts with the retinitis pigmentosa protein 2 (RP2), a plasma membrane-associated protein whose molecular function remains to be identified.24 Mutations in RP2 cause X-linked retinitis pigmentosa (XLRP), a genetically heterogeneous disease resulting in severe retinal degeneration.25 Recombinant Arl3 also interacts with the delta subunit of rod-specific cyclic GMP phosphodiesterase, a key effector molecule in visual transduction.48

The most striking features of the retina in mice lacking Arl3 are the abundance of TUNEL-positive photoreceptors and rhodopsin mislocalization in rod cell bodies. These defects are not attributable to a lack of connecting cilia in photoreceptors because basal bodies and connecting cilia are discernible at the ultrastructural level in mice deficient in Arl3. Rhodopsin synthesis and transport to the OS is an area of intense investigation and the fact that mutations in the rhodopsin gene that affect trafficking to OSs cause retinitis pigmentosa in humans.49 Rhodopsin is packed in vesicles and transported from the Golgi apparatus to the distal end of the IS. Rhodopsin vesicles appear to fuse with the plasma membrane at the level of the basal bodies. Genetic evidence and immunoelectron microscopy have revealed several proteins involved in active transport of opsins from the IS to the OS.50 Rhodopsin mislocalization to cell bodies implies that Arl3 may facilitate transport of this photopigment from the Golgi apparatus to the connecting cilia.

Some of the Arls have been localized to the Golgi apparatus, others such as Arl3 have been associated with microtubules and mutations in related proteins in several species result in abnormal vesicle trafficking. Photoreceptor disks and visual pigments are transported via the connecting cilium (photosensory primary cilia) at a rapid rate, and this transportation is dependent on IFT.17 This study does not address the precise cellular function of Arl3, but in light of the mislocalization of rhodopsin, we speculate that Arl3 regulates the trafficking of proteins critical for the function and maintenance of photoreceptor OSs. Although only rhodopsin was studied here, it is likely that disruption of Arl3 function impairs transport of other proteins into rods and cones as well. Nevertheless, the lack of obvious OSs and abnormally shortened ISs in our findings demonstrates that Arl3 is required for photoreceptor development and survival.

Our data indicate that Arl3 is important in renal, liver, and pancreatic function as well as photoreceptor development. We have presented here a mouse model with PKD and photoreceptor defects that we suggest are attributable to abrogated mechanosensory and photosensory cilia function. The presence of apparently normal cilia in the Arl3 (−/−) mice suggests that the primary defect is not structurally defective cilia, but that Arl3 function may be critical for proper regulation of the intracellular transport process and/or Ca++ signaling and proliferation. The Arl3 (−/−) knockout mouse model provides a useful tool for further studies of PKD, photoreceptor development, and the potential role of primary ciliary function in a wide variety of disease states.

Acknowledgments

We thank Drs. Alex Turner, Liz Richter, and the external reviewers for their thoughtful suggestions and critical reading of this manuscript; and Laura Kirkham and Jeni Hyland for their careful attention to animal care and breeding.

Footnotes

Address reprint requests to Jeffrey J. Schrick, Lexicon Genetics Inc., 8800 Technology Forest Pl., The Woodlands, TX 77381. .moc.negxel@kcirhcsj :liam-E

Supported by Lexicon Genetics, Incorporated.

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