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.

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 for the (+/+) allele. The LTR2 primer, complementary to the gene-trapping vector, and primer B amplify the mutated allele. C: Arl3 RT-PCR. Primers C and D are complementary to Arl3 exons 1 and 2 flanking the insertion site of the gene-trapping vector. RT-PCR using primers C and D shows absence of endogenous message in the kidney and spleen of (−/−) animals. RT, reverse transcription.

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 Arl3 (+/+) mouse is shown for comparison. D: In the Arl3 (−/−) mouse, the pronounced distention of the flanks was caused by severe bilateral renomegaly (1.2 × 1 × 1 cm, white arrows). The kidneys were uniformly enlarged by innumerable translucent and minute cystic structures throughout the organ. The pancreatic ducts, gall bladder, and bile ducts were enlarged and tortuous (red arrows).

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 tubules of the kidney. Note the relative absence of staining in the medulla and papilla. The inset shows at higher magnification the marked difference in cortical and medullary staining. B: In (−/−) mice, there are myriad fluid-filled cysts in both the cortex and medulla. These cysts involve all segments of the nephron, including proximal and distal tubules, collecting ducts, and even glomerular Bowman’s spaces (black arrows). C: In Arl3 (−/−) mice, numerous proliferating cells are present in cystic collecting ducts as well as cortical tubules as shown by Ki-67 staining (brown). D: Prominent lesions in the pancreas of Arl3 knockout mice include widespread cystic dilatation of intralobular pancreatic ducts accompanied by atrophy and loss of exocrine pancreas tissue. Note the disorganized, small, irregular nests of exocrine cells surrounded by loose interstitial tissues (red arrows). E: In many cystic pancreatic ducts, more than 80% of the epithelial cells were Ki-67-positive. F: Scattered individual cells lining the cystic pancreatic ducts were positive for insulin by immunohistochemistry. G: Liver section from Arl3 (+/+) mice shows the normal diameter of intrahepatic bile ducts. H: In Arl3 (−/−) mice, the intrahepatic bile ducts were often distorted by multiple segmental and saccular dilatations. Scale bars, 100 μm (E, G, and H).

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 layer). Photoreceptors are located in the ONL and their inner segments (IS) and outer segments (OS) are located directly beneath the retinal pigment epithelium (RPE). B: Three cell layers are apparent in mice lacking Arl3. However, the region containing the OS and IS appears shortened at this age. C: Electron microscopy of (+/+) outer retina at postnatal day 9. Photoreceptor ISs are located above the outer limiting membrane (OLM) and contain basal bodies (arrow). Connecting cilia (arrowheads) are also prominent at this age. Developing OS disks (asterisks) are located beneath the retinal pigment epithelium (RPE). Scale bar represents 50 μm in A, B and 2 μm in C, D. D:Mice lacking Arl₃ exhibit a paucity of OS disks, although basal bodies (arrow) are present.

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 OS and IS located beneath the retinal pigment epithelium are intensely labeled with PNA. In contrast, only cone ISs are apparent in mice lacking Arl3. C and D: Rods were labeled with an antibody specific for rhodopsin. C: In (+/+) retinas, strong rhodopsin immunoreactivity is present in OSs located directly above the IS. This reactivity corresponds to the rod OS disks. D: (−/−) animals failed to shown specific OS staining. Rod photoreceptor cell bodies were strongly reactive for rhodopsin antibodies, indicating an accumulation of this photopigment in the absence of Arl3. Scale bar, 75 μm (B).

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 INL at this age. These cells represent naturally occurring cell death during retinal development. C: Nomarski image of the (−/−) retina shown in D. D: Many TUNEL-positive cells (arrowheads) are located in the ONL in the absence of Arl3. TUNEL-positive cells in the INL (arrows) are also present, but their density appears similar to the (+/+) shown in B. Scale bar, 100 μm (D).