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Modelling a ciliopathy: Ahi1 knockdown in model systems reveals an essential role in brain, retinal, and renal development

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Abstract

Joubert syndrome and related diseases (JSRD) are cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy, and nephronophthisis (a cystic kidney disease). Mutations in AHI1 are the most common genetic cause of JSRD, with developmental hindbrain anomalies and retinal degeneration being prominent features. We demonstrate that Ahi1, a WD40 domain-containing protein, is highly conserved throughout evolution and its expression associates with ciliated organisms. In zebrafish ahi1 morphants, the phenotypic spectrum of JSRD is modeled, with embryos showing brain, eye, and ear abnormalities, together with renal cysts and cloacal dilatation. Following ahi1 knockdown in zebrafish, we demonstrate loss of cilia at Kupffer’s vesicle and subsequently defects in cardiac left–right asymmetry. Finally, using siRNA in renal epithelial cells we demonstrate a role for Ahi1 in both ciliogenesis and cell–cell junction formation. These data support a role for Ahi1 in epithelial cell organization and ciliary formation and explain the ciliopathy phenotype of AHI1 mutations in man.

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Acknowledgments

We would like to thank Mike Shaw (University of Oxford) for assistance with scanning EM, David Studholme (University of Exeter) for assistance with bioinformatics, and Keith Gull (University of Oxford) for many helpful discussions. We are extremely grateful to Kidney Research UK and the Medical Research Council (Training Fellowship to RJS) and the Mason Medical Research Fellowship (for pump priming funding to RJS). We also acknowledge support from the Northern Counties Kidney Research Fund and Newcastle Hospitals Healthcare Charity (support for AMH), the Kids Kidney Research Fund (support for LE), the Beit Memorial Fellowships for Medical Research (to HRD), the EP Abraham Trust, and GlaxoSmithKline (Clinician Scientist Fellowship to JAS) for funding.

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Authors and Affiliations

  1. Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK

    Roslyn J. Simms, Ann Marie Hynes, Lorraine Eley, Bill Chaudhry & John A. Sayer

  2. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

    David Inglis

  3. Biosciences: College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK

    Helen R. Dawe

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  1. Roslyn J. Simms
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Correspondence to John A. Sayer.

Electronic supplementary material

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18_2011_826_MOESM1_ESM.tif

Supplementary Figure 1. Ahi1 protein domains in man, mouse, and zebrafish. Domain structure of the Ahi1 proteins from human mouse and zebrafish. Human Ahi1 (alias Jouberin) is a 1,196-amino acid (aa) protein that contains a Src-homology 3 domain (SH3), six WD40 domains (WD40), and an N-terminal coiled-coil (CC) domain. The mouse Ahi1 is 1,047 aa protein that lacks the N-terminal CC domain and has seven WD40 domains. The zebrafish ahi1 is a 934-aa protein with the predicted protein domains conserved. (TIFF 620 kb)

18_2011_826_MOESM2_ESM.tif

Supplementary Figure 2. Negative and positive controls for whole mount zebrafish in situ hybridization experiments. Negative controls for in situ expression in the developing zebrafish: Whole-mount zebrafish embryos at (A) 12–14 hpf (6–10 somites), (B, C) 24 hpf, (D, E) 48 hpf, and (F) 72 hpf with omission of ahi1 antisense riboprobe. Whole-mount zebrafish embryos at (G) 12–14 hpf (6–10 somites), (H and I) 24 hpf with ahi1 sense riboprobe. Positive controls for in situ expression in the developing zebrafish: A shh riboprobe provided a positive control and confirms characteristic expression (J–L). shh is expressed in the notochord and neuroectoderm at 12–14 hpf (arrowed in J), distinctly in the diencephalon at 24 hpf (arrowed in K) and in the branchial arches at 48 hpf (arrowed in L). Scale bar = 100 μm. (TIFF 3946 kb)

18_2011_826_MOESM3_ESM.tif

Supplementary Figure 3. Disrupted retinal development in ahi1 morphants. Eye histology in uninjected WT control and ahi1 MO-injected embryos at 72–120 hpf. (A, B) Control embryos show normal retinal lamination with three cell layers (GL, INL, and ONL). (C, D) In ahi1 SPL8 MO-injected embryos there is a severe retinal phenotype, retinal lamination does not occur and distinction of the three layers (GL, INL, and ONL) is not possible. The retinal pigment epithelium (RPE, arrowhead) was present in the ahi1 morphants. (GL ganglion cell layer; INL inner nuclear layer; ONL outer nuclear layer). Scale bar = 50 μm. (TIFF 3873 kb)

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Supplementary Figure 4. Quantification of abnormal phenotypes in ahi1 MO-injected embryos and dose-dependant mortality of ahi1 MO injection. Quantification of the frequency of abnormal phenotypes in ahi1 MO-injected embryos. None of the anomalies were noted in uninjected control embryos. (A) Percentage of abnormal phenotypes are shown as mean ± SEM following ahi1 ATG MO-injection of embryos (using 6-ng and 3-ng doses, total number of embryos injected = 303). (B) Percentage of abnormal phenotypes are shown as mean ± SEM following ahi1 SPL8 MO-injected (using 2-ng and 1-ng doses, total number of embryos injected = 1,044). (C) Dose-dependant effect on mortality at 24 hpf is shown as mean % ± SEM using a dose range of 1 to 6 ng of ahi1 SPL8 MO. (D) Quantification of rescue of combined phenotype following injection of ahi1 SPL8 MO (2-ng dose) with Ahi1 mRNA compared to ahi1 SPL8 MO alone. There was a significant degree of rescue to uninjected wild-type phenotype (*, p < 0.0001, Fisher’s exact test). (E) Quantification of rescue of each variant of phenotype following injection of ahi1 SPL8 MO (2-ng dose) with Ahi1 mRNA compared to ahi1 SPL8 MO alone. Comparing treatments for each phenotype there was a significant rescue of wild-type phenotype and a significant reduction in disease phenotypes (curly tail, cardiac edema, hydocephalus, renal cyst, and otic placode abnormality) following ahi1 MO + mRNA injection (*, p < 0.0001, Fisher’s exact test). (TIFF 1446 kb)

18_2011_826_MOESM5_ESM.tif

Supplementary Figure 5. Morphology of KV in ahi1 morphants is preserved. Light microscopy images of 8–10 somite stage embryos during development and following in situ hybridization using a probe directed toward charon demonstrate preserved KV in ahi1 morphants. Lateral (A) and dorsal view (B) of control embryos showing KV (arrow). (C) A charon in situ hybridization probe identifies KV in a control embryo (yolk sac removed). Lateral (D) and dorsal views (E) of ahi1 morphant showing KV is intact. (F) A charon in situ hybridization probe identifies KV in a morphant embryo (yolk sac removed). (TIFF 6693 kb)

18_2011_826_MOESM6_ESM.tif

Supplementary Figure 6. Analysis of Kupffer’s vesicle (KV) and cardiac looping phenotypes in ahi1 MO-injected embryos (A). Quantification of the frequency of KV phenotypes in ahi1 SPL8 MO-injected embryos compared to uninjected controls. There is a significant reduction in ciliated KV (*, p < 0.0001, Fisher’s exact test) following 1–2 ng ahi1 SPL8 MO injection. (B) Quantification of the frequency of cardiac looping phenotypes in ahi1 SPL8 MO-injected embryos compared to uninjected controls. There is a significant reduction in normal cardiac D-looping (*, p < 0.0001, Fisher’s exact test) following 2-ng ahi1 SPL8 MO injection. The majority of embryos show a reversal of cardiac asymmetry (L-looping). (TIFF 249 kb)

18_2011_826_MOESM7_ESM.tif

Supplementary Table 1. Distribution of Ahi1 proteins and cilium and centriole architecture across eukaryotes. A putative Ahi1 homologue is found in organisms that build both motile and sensory cilia, but only if a triplet centriole architecture is also present. Ultrastructural information was not available for all organisms included in this study. A ? denotes an unknown architecture or one where conflicting data have been reported. In most cases, architectures are in accordance with those described in Woodland & Fry [64]. (TIFF 1399 kb)

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Simms, R.J., Hynes, A.M., Eley, L. et al. Modelling a ciliopathy: Ahi1 knockdown in model systems reveals an essential role in brain, retinal, and renal development. Cell. Mol. Life Sci. 69, 993–1009 (2012). https://doi.org/10.1007/s00018-011-0826-z

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