C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes

Etude de la fonction du gène rpgrip1l dans les processus de différenciation et de polarité cellulaire chez le poisson-zèbre

Le cil primaire, présent à la surface de la majorité des cellules chez les vertébrés, a un rôle primordial dans le développement embryonnaire et dans la modulation de voies de signalisation, notamment Hedgehog et Wnt. Des défauts de cette structure sont associés à des maladies héréditaires, les ciliopathies. Le gène Rpgrip1l est impliqué dans deux ciliopathies, le syndrome de Joubert de type B et le syndrome de Meckel, caractérisées par une polydactylie, des kystes rénaux et des malformations du système nerveux central. Rpgrip1l code pour une protéine localisée principalement à la zone de transition des cils, une région impliquée dans le contrôle du trafic des protéines vers et hors du compartiment ciliaire. Mon projet de thèse a porté sur les fonctions de Rpgrip1l dans les processus de différenciation et de polarité cellulaire dans le système nerveux du poisson-zèbre. Mon premier travail, basé sur la perte de fonction de rpgrip1l par injection de morpholino dans des embryons de poissons zèbre, a mis à jour une fonction de Rpgrip1l dans la mise en place de la polarité planaire via la stabilisation de dishevelled, une protéine clé de la voie Wnt/PCP. J ai ensuite étudié les fonctions plus tardives du gène rpgrip1l et de son paralogue rpgrip1 dans la rétine, via l analyse de lignées de poissons zèbre hypomorphes. Cette étude a montré l existence de multiples isoformes de Rpgrip1l et suggère de nouvelles fonctions extra-ciliaires pour Rpgrip1l dans la morphogenèse des photorécepteurs. L ensemble de ce travail devrait permettre de mieux comprendre l origine développementale des anomalies cérébrales et rétiniennes observées dans les ciliopathies.In vertebrates, primary cilia are present in virtually every cell and are involved in several signaling pathways such as the Hedgehog and Wnt pathways. Cilium dysfunctions have been causally linked to a group of pleiotropic and genetically heterogeneous human diseases, the ciliopathies. The human RPGRIP1L gene is one of the causal genes in Meckel and Joubert type B syndromes, two ciliopathies characterized by polydactyly, kidney cysts, and central nervous system malformations. The Rpgrip1l protein is enriched in the ciliary transition zone that establishes the ciliary gate controlling entry and exit of proteins in and out of the cilium. During my PhD, I studied the function of the Rpgrip1l gene in differentiation and planar cell polarity events that participate in brain morphogenesis. The first study, based on the loss of function of rpgrip1l by morpholino injection in zebrafish embryo, highlights rpgrip1l function in planar cell polarity via dishevelled stabilization, a core protein of Wnt/PCP pathway. Then, I studied the later functions of rpgrip1l and its paralogue rpgrip1 in the retina, using hypomorphic zebrafish. This work, by the discovery of rpgrip1l variants and description of photoreceptors defects, provides leads for new extra-ciliary functions of rpgrip1l in retinal morphogenesis. Ultimately, this work should result in a better understanding of the developmental origin of cerebral and retinal defects found in ciliopathies.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Functional analysis of the transition zone protein Rpgrip1l during zebrafish development

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Rpgrip1l controls ciliary gating by ensuring the proper amount of Cep290 at the vertebrate transition zone

A range of severe human diseases called ciliopathies is caused by the dysfunction of primary cilia. Primary cilia are cytoplasmic protrusions consisting of the basal body (BB), the axoneme, and the transition zone (TZ). The BB is a modified mother centriole from which the axoneme, the microtubule-based ciliary scaffold, is formed. At the proximal end of the axoneme, the TZ functions as the ciliary gate governing ciliary protein entry and exit. Since ciliopathies often develop due to mutations in genes encoding proteins that localize to the TZ, the understanding of the mechanisms underlying TZ function is of eminent importance. Here, we show that the ciliopathy protein Rpgrip1l governs ciliary gating by ensuring the proper amount of Cep290 at the vertebrate TZ. Further, we identified the flavonoid eupatilin as a potential agent to tackle ciliopathies caused by mutations in RPGRIP1L as it rescues ciliary gating in the absence of Rpgrip1l

The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals

International audienceCilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates

Astrogliosis and Neuroinflammation Underlie Scoliosis Upon Cilia Dysfunction

Cilia defects lead to scoliosis in zebrafish, but the underlying pathogenic mechanisms are poorly understood and may diverge depending on the mutated gene. We dissected the mechanisms of scoliosis onset in a zebrafish mutant for the rpgrip1l gene encoding a ciliary transition zone protein. rpgrip1l mutant fish developed scoliosis with near-total penetrance but asynchronous onset in juveniles. Taking advantage of this asynchrony, we found that curvature onset was preceded by brain ventricle dilations and concomitant to the perturbation of Reissner fiber polymerization and to the loss of multicilia tufts around the subcommissural organ. Rescue experiments showed that Rpgrip1l was exclusively required in foxj1a-expressing cells to prevent axis curvature. Transcriptomic and proteomic studies identified neuroinflammation associated with increased Annexin levels as a potential mechanism of scoliosis development in rpgrip1l juveniles. Investigating the cell types associated with annexin2 over-expression, we uncovered astrogliosis, arising in glial cells surrounding the diencephalic and rhombencephalic ventricles just before scoliosis onset and increasing with time in severity. Anti-inflammatory drug treatment reduced scoliosis penetrance and severity and this correlated with both reduced astrogliosis and macrophage/microglia enrichment around the diencephalic ventricle. Mutation of the cep290 gene encoding another transition zone protein also associated astrogliosis with scoliosis. Thus, we propose that the onset of a feed-forward loop between astrogliosis, induced by perturbed ventricular homeostasis, and immune cells recruitment as a novel pathogenic mechanism of zebrafish scoliosis in ciliary transition zone mutants

Multi-fonctional role of the Krox-20 regulatory gene: from specification of positional information to cell differentiation

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[Role of the Krox-20 gene in the development of rhombencephalon].

International audienceIn the hindbrain region of the developing CNS, anteroposterior patterning involves a transient segmentation process which leads to the formation of morphological bulges called rhombomeres. The rhombomeres constitute cell lineage restriction units and participate in the establishment of a metameric pattern which is responsible for the segmental organisation of motor and reticular neurons. Like Drosophila compartments, rhombomeres also constitute domains of specific gene expression. Genes expressed in a rhombomere-specific manner so far identified encode various types of putative regulatory molecules, including transcription factors, like Hox proteins, the zinc finger protein Krox-20 and the basic domain leucine-zipper protein kreisler, and receptor type molecules, like Sek-1, a member of the EPH family of tyrosine kinase receptors. Such genes are thought to play a role either in the definition of segmental territories or in the specification of the identity of the rhombomeres. Initial analysis of the function of some of these genes have indeed supported this hypothesis. This is the case for the Krox-20 gene. It is expressed within the developing hindbrain in two transverse domains which prefigure and then coincide with r3 and r5. We have inactivated Krox-20 by homologous recombination in ES cells and demonstrated that the mutation leads to the deletion of r3 and r5. The mutation introduced into the Krox-20 gene involved the in-frame insertion of the lacZ coding sequence. This allowed us to follow the late expression pattern of the gene and to identify two additional phenotypes, affecting myelination of the peripheral nervous system and endochondral ossification. The lacZ reporter also permitted a detailed analysis of the expression of Krox-20 in peripheral glial cells, revealing important steps in the control of their development. Recently we have performed a detailed analysis of specific neuronal populations affected by the mutation which shed new light on the role of Krox-20 in the segmentation and on the physiological consequences of its inactivation. We have also identified several new members of the Sek-1 family of receptor tyrosine kinases, which are also expressed in a rhombomere-specific manner. Finally, we have provided evidence that Krox-20 is as a key regulator of r3/r5-specific transcription, controlling the expression of at least five other regulator genes in these rhombomeres. In three cases, Hoxb-2, Hoxa-2 and Sek-1, we could demonstrate that Krox-20 was directly involved in the transcriptional activation of these genes

Dishevelled stablisation at the cilium by RPGRIP1L is essential for planar cell polarity

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