Hippocampals neurogenesis is impaired in mice with a deletion in the coiled coil domain of Talpid3-implications for Joubert syndrome.

Mutations in Talpid3, a basal body protein essential for the assembly of primary cilia, have been reported to be causative for Joubert Syndrome (JS). Herein, we report prominent developmental defects in the hippocampus of a conditional knockout mouse lacking the conserved exons 11 and 12 of Talpid3. At early postnatal stages, the Talpid3 mutants exhibit a reduction in proliferation in the dentate gyrus and a disrupted glial scaffold. The occurrence of mis-localized progenitors in the granule cell layer suggests a role for the disrupted glial scaffold in cell migration resulting in defective subpial neurogenic zone-to-hilar transition. Neurospheres derived from the hippocampus of Talpid3fl/flUbcCre mouse, in which Talpid3 was conditionally deleted, lacked primary cilia and were smaller in size. In addition, neurosphere cells showed a disrupted actin cytoskeleton and defective migration. Our findings suggest a link between the hippocampal defects and the learning/memory deficits seen in JS patients.

Mice with a conditional deletion of Talpid3 (KIAA0586) - a model for Joubert syndrome.

Joubert syndrome (JS) is a ciliopathy associated with mutations in numerous genes encoding cilia components. TALPID3 encoded by KIAA0856 in man (2700049A03Rik in mouse) is a centrosomal protein essential for the assembly of primary cilia. Mutations in KIAA0856 have been recently identified in JS patients. Herein, we describe a novel mouse JS model with a conditional deletion of the conserved exons 11-12 of Talpid3 in the central nervous system which recapitulates the complete cerebellar phenotype seen in JS. Talpid3 mutant mice exhibit key hallmarks of JS including progressive ataxia, severely hypoplastic cerebellar hemispheres and vermis, together with abnormal decussation of the superior cerebellar peduncles. The Purkinje cell layer is disorganised with abnormal dendritic arborisation. The external granule layer (EGL) is thinner, lacks primary cilia, and has a reduced level of proliferation. Furthermore, we describe novel cellular defects including ectopic clusters of mature granule neurons, and abnormal parallel fibre-derived synapses and disorientation of cells in the EGL. The defective glial scaffold results in abnormal granule cell migration which manifests as ectopic clusters of granule neurons. In addition, we show a reduction in Wnt7a expression suggesting that defects may arise not only from deficiencies in the Hedgehog (Hh) pathway but also due to the additional roles of Talpid3. The Talpid3 conditional knockout mouse is a novel JS model which fully recapitulates the JS cerebellar phenotype. These findings reveal a role for Talpid3 in granule precursor cell migration in the cerebellum (either direct or indirect) which together with defective Hh signalling underlies the JS phenotype. Our findings also illustrate the utility of creating conditional mouse models to assist in unravelling the molecular and cellular mechanisms underlying JS. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Expression of glycosaminoglycan epitopes during zebrafish skeletogenesis.

BACKGROUND: The zebrafish is an important developmental model. Surprisingly, there are few studies that describe the glycosaminoglycan composition of its extracellular matrix during skeletogenesis. Glycosaminoglycans on proteoglycans contribute to the material properties of musculo skeletal connective tissues, and are important in regulating signalling events during morphogenesis. Sulfation motifs within the chain structure of glycosaminoglycans on cell-associated and extracellular matrix proteoglycans allow them to bind and regulate the sequestration/presentation of bioactive signalling molecules important in musculo-skeletal development. RESULTS: We describe the spatio-temporal expression of different glycosaminoglycan moieties during zebrafish skeletogenesis with antibodies recognising (1) native sulfation motifs within chondroitin and keratan sulfate chains, and (2) enzyme-generated neoepitope sequences within the chain structure of chondroitin sulfate (i.e., 0-, 4-, and 6-sulfated isoforms) and heparan sulfate glycosaminoglycans. We show that all the glycosaminoglycan moieties investigated are expressed within the developing skeletal tissues of larval zebrafish. However, subtle changes in their patterns of spatio-temporal expression over the period examined suggest that their expression is tightly and dynamically controlled during development. CONCLUSIONS: The subtle differences observed in the domains of expression between different glycosaminoglycan moieties suggest differences in their functional roles during establishment of the primitive analogues of the skeleton.

Spinal deformity in aged zebrafish is accompanied by degenerative changes to their vertebrae that resemble osteoarthritis.

Age-related degenerative changes within the vertebral column are a significant cause of morbidity with considerable socio-economic impact worldwide. An improved understanding of these changes through the development of experimental models may lead to improvements in existing clinical treatment options. The zebrafish is a well-established model for the study of skeletogenesis with significant potential in gerontological research. With advancing age, zebrafish frequently develop gross deformities of their vertebral column, previously ascribed to reduced trunk muscle tone. In this study, we assess degenerative changes specifically within the bone and cartilage of the vertebral column of zebrafish at 1, 2 and 3-years of age. We show increased frequency and severity of spinal deformities/curvatures with age. Underlying the most severe phenotypes are partial or complete vertebral dislocations and focal thickening of the vertebral bone at the joint margins. MicroCT examination demonstrates small defects, fractures and morphological evidence suggestive of bone erosion and remodeling (i.e. osteophytes) within the vertebrae during aging, but no significant change in bone density. Light and electron microscopic examination reveal striking age-related changes in cell morphology, suggestive of chondroptosis, and tissue remodelling of the vertebral cartilage, particularly within the pericellular micro-environment. Glycosaminoglycan analysis of the vertebral column by HPLC demonstrates a consistent, age-related increase in the yield of total chondroitin sulfate disaccharide, but no change in sulfation pattern, supported by immunohistochemical analysis. Immunohistochemistry strongly identifies all three chondroitin/dermatan sulphate isoforms (C-0-S, C-4-S/DS and C-6-S) within the vertebral cartilage, particularly within the pericellular micro-environment. In contrast, keratan sulfate immunolocalises specifically with the notochordal tissue of the intervertebral disc, and its labelling diminishes with age. In summary, these observations raise the prospect that zebrafish, in addition to modelling skeletal development, may have utility in modelling age-related degenerative changes that affect the skeleton during senescence.