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Front Physiol
2019 Jan 01;10:134. doi: 10.3389/fphys.2019.00134.
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The Frog Xenopus as a Model to Study Joubert Syndrome: The Case of a Human Patient With Compound Heterozygous Variants in PIBF1.
Ott T
,
Kaufmann L
,
Granzow M
,
Hinderhofer K
,
Bartram CR
,
Theiß S
,
Seitz A
,
Paramasivam N
,
Schulz A
,
Moog U
,
Blum M
,
Evers CM
.
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Joubert syndrome (JS) is a congenital autosomal-recessive or-in rare cases-X-linked inherited disease. The diagnostic hallmark of the so-called molar tooth sign describes the morphological manifestation of the mid- and hind-brain in axial brain scans. Affected individuals show delayed development, intellectual disability, ataxia, hyperpnea, sleep apnea, abnormal eye, and tongue movements as well as hypotonia. At the cellular level, JS is associated with the compromised biogenesis of sensory cilia, which identifies JS as a member of the large group of ciliopathies. Here we report on the identification of novel compound heterozygous variants (p.Y503C and p.Q485*) in the centrosomal gene PIBF1 in a patient with JS via trio whole exome sequencing. We have studied the underlying disease mechanism in the frog Xenopus, which offers fast assessment of cilia functions in a number of embryological contexts. Morpholino oligomer (MO) mediated knockdown of the orthologous Xenopus pibf1 gene resulted in defective mucociliary clearance in the larval epidermis, due to reduced cilia numbers and motility on multiciliated cells. To functionally assess patient alleles, mutations were analyzed in the larval skin: the p.Q485* nonsense mutation resulted in a disturbed localization of PIBF1 to the ciliary base. This mutant failed to rescue the ciliation phenotype following knockdown of endogenous pibf1. In contrast, the missense variant p.Y503C resulted in attenuated rescue capacity compared to the wild type allele. Based on these results, we conclude that in the case of this patient, JS is the result of a pathogenic combination of an amorphic and a hypomorphic PIBF1 allele. Our study underscores the versatility of the Xenopus model to study ciliopathies such as JS in a rapid and cost-effective manner, which should render this animal model attractive for future studies of human ciliopathies.
Figure 1. Pedigree and MRI scans. (A) Pedigree of patient family. Black symbol, affected individual; White symbols, unaffected individuals. (BâH) MR imaging of the patient at age 2 years and 6 months. Sagittal (B) and axial (CâH) images showed polymicrogyria in the parietal and temporal region (C,D) and hypoplasia of vermis cerebellum (B,EâH). Axial MR images of cerebellum and brainstem (EâH) showed a mild âmolar tooth signâ (marked with white arrows in FâH) due to a deep interpeduncular fossa, prominent and elongated superior cerebellar peduncles and a hypoplastic cerebellar vermis. (IâL) Corresponding MR images of a healthy control
Figure 2. PIBF1 protein conservation and domain structure. (A) Sanger sequencing of PIBF1 from RT-PCR product of the patient mRNA extracted from blood. Black arrow heads indicate position of mutations. (B) Conservation of amino acid sequences between human, mouse, Xenopus, and zebrafish PIBF1. (C) Putative domain structure. JS and microcephaly (MC) mutations are indicated as red bars. For details see text. *Novel mutations identified in this study.
Figure 3. Embryonic pibf1 expression correlates with ciliated tissues. Embryos of defined stages were analyzed for pibf1 mRNA by whole-mount in situ hybridization using a digoxigenin-labeled antisense probe. (A) Maternal transcripts in the animal hemisphere of the 4-cell embryo. (B) Expression in the involuting marginal zone tissue of the early gastrulaembryo. (C) Staining of pibf1 in the axial and paraxial mesoderm at early neurula stages. (D) Pibf1 signals in skin MCCs and axial/paraxial mesoderm. (E) Expression in the otic vesicle at stage 25. (F–H) Expression pattern in tadpoles, including the head region, somites, and nephrostomes. Histological section of the eye (H′) revealed signals in the retina as well as the inner nuclear cell layer. Planes of histological sections in (B′,D′,E′,F′,H′) are indicated in the respective panels. an, animal; a, anterior; d, dorsal; DL, dorsal lip; INL, inner nuclear layer; l, left; neph, nephrostomes; no, notochord; nt, neural tube; ov, otic vesicle; p, posterior; r, right; re, retina; som, somite; v, ventral; veg, vegetal.
Figure 4. Pibf1 localization to MCC basal bodies is attenuated in nonsense JS variant. (A–C) Localization of the endogenous Pibf1 protein, using an anti-PIBF1 antibody (A) and co-staining with Centrin 1 (B) and Hoechst 33342 to highlight the nucleus (C). (D–F) An EGFP-PIBF1 fusion protein recapitulates the staining of the endogenous protein. (G–I) Unaltered expression of the missense allele Y503C. (J–L) Attenuated basal body localization of the truncated Q485* variant of PIBF1. Arrowheads mark protein aggregates observed upon overexpression of EGFP fusion
Figure 5. Loss of Pibf1 protein in pibf1 morphant Xenopus skin MCCs. Basal body staining of Pibf1 (A–C) was lost in TBMO injected specimen (D–F). Tjp1 immunofluorescence was used to mark cell boundaries (B,C,E,F). Fluorescent dextrane (green) was co-injected as lineage tracer to control targeting of injections (E,F). Arrowheads highlight MCCcilia in WT embryo.
Figure 6. Rescue of morphant MCC ciliation is lost with mutant PIBF1 alleles. (A–E) MCC ciliation in WT embryos was lost in pibf1 TBMO-injected specimens. (E–G) Rescue of ciliation upon co-injection of TBMO and EGFP-PIBF1. (E,H,I) Attenuated rescue in embryos co-injected with missense PIBF1 allele. (E,J,K) Defective rescue upon co-injection of the nonsense PIBF1 allele. (E) Quantification of results. (L,M) Kymographs representing ciliary beating in WT and TBMO-injected stage 30 larval skin MCCs. ***Very highly significant, p < 0.01.
pibf1 (progesterone immunomodulatory binding factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, horizontal view, animal up.
pibf1 (progesterone immunomodulatory binding factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
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