Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo

Functional characterization of two new ciliary genes during the development of vertebrate

Les cils et les flagelles sont des organites cellulaires très conservés qui assurent des fonctions essentielles. Chez l'Homme, les défauts d'assemblage des cils et des flagelles conduisent à de multiples pathologies, les ciliopathies. Afin de comprendre comment se forment et fonctionnent les cils, j'ai analysé la fonction de deux nouveaux gènes identifiés comme cible des facteurs de transcription de ciliogenèse RFX. Tout d'abord je me suis focalisée sur le gène CCDC151, évolutivement conservé dans les espèces possédant des cils motiles. J'ai pu montrer que CCDC151 est impliquée dans le transport dépendant de l'IFT des bras de dynéine chez les animaux et qu'elle est nécessaire à la perception sensorielle chez la drosophile. Par ailleurs, j'ai également montré que cette protéine possède des fonctions cellulaires additionnelles puisqu'elle est requise pour l'orientation correcte des plans de division cellulaire et qu'elle est impliquée dans la régulation de la taille du cil primaire chez les mammifères. Je me suis ensuite intéressée au gène LRRC48 également conservée dans les espèces possédant des cils motiles. Cette protéine est nécessaire à la motilité des flagelles de spermatozoïdes et des cils des neurones sensoriels en 9+0 et dans la réponse auditive chez la drosophile. De plus LRRC48 est indispensable au développement des vertébrés puisque son absence chez le poisson zèbre conduit à l'hydrocéphalie, des kystes rénaux et des défauts de motilité des cils. Elle est également essentielle à la biogenèse de l'oreille dans cet organisme.En conclusion, il s'agit de deux nouveaux acteurs de la ciliogenèse potentiellement impliqués dans les pathologies ciliaires chez l'HommeCilia are highly conserved structures found from protozoa to mammals where they play essential physiological and developmental functions and cilia dysfunction leads to various syndromes in humans known as ciliopathies. To understand cilia formation and function, I performed functional analysis of two new target genes of the RFX ciliogenic transcription factors. First, I focused on CCDC151 that is evolutionary conserved in motile ciliated species. I showed that CCDC151 is involved in the control of IFT-dependent dynein arm assembly in animals and required for geotaxis behavior of adult flies. In zebrafish, depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts, two phenotypes resulting from impaired ciliary beating. However, I also showed that CCDC151 is also implicated in other cellular functions in vertebrates as it is involved in proper orientation of cell divisions and implicated in the regulation of primary cilium length in mammalian cells. In a second part, I studied LRRC48 that is also conserved in species with motile cilia. I showed that this protein is essential for motility of flagellar spermatozoids and for motility of the 9+0 sensory cilia as well as in the auditory response in drosophila. In zebrafish, morpholinos induced depletion of this protein leads to hydrocephaly, kidney cysts, inner ear abnormalities and cilia motility defects. Moreover this protein is also required for inner ear biogenesis in the model. In conclusion, these two genes are essential for ciliogenesis and they are new candidate genes potentially implicated in human ciliary disease

Caractérisation fonctionnelle de deux nouveaux gènes ciliaires pendant le développement des vertébrés

Cilia are highly conserved structures found from protozoa to mammals where they play essential physiological and developmental functions and cilia dysfunction leads to various syndromes in humans known as ciliopathies. To understand cilia formation and function, I performed functional analysis of two new target genes of the RFX ciliogenic transcription factors. First, I focused on CCDC151 that is evolutionary conserved in motile ciliated species. I showed that CCDC151 is involved in the control of IFT-dependent dynein arm assembly in animals and required for geotaxis behavior of adult flies. In zebrafish, depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts, two phenotypes resulting from impaired ciliary beating. However, I also showed that CCDC151 is also implicated in other cellular functions in vertebrates as it is involved in proper orientation of cell divisions and implicated in the regulation of primary cilium length in mammalian cells. In a second part, I studied LRRC48 that is also conserved in species with motile cilia. I showed that this protein is essential for motility of flagellar spermatozoids and for motility of the 9+0 sensory cilia as well as in the auditory response in drosophila. In zebrafish, morpholinos induced depletion of this protein leads to hydrocephaly, kidney cysts, inner ear abnormalities and cilia motility defects. Moreover this protein is also required for inner ear biogenesis in the model. In conclusion, these two genes are essential for ciliogenesis and they are new candidate genes potentially implicated in human ciliary diseasesLes cils et les flagelles sont des organites cellulaires très conservés qui assurent des fonctions essentielles. Chez l'Homme, les défauts d'assemblage des cils et des flagelles conduisent à de multiples pathologies, les ciliopathies. Afin de comprendre comment se forment et fonctionnent les cils, j'ai analysé la fonction de deux nouveaux gènes identifiés comme cible des facteurs de transcription de ciliogenèse RFX. Tout d'abord je me suis focalisée sur le gène CCDC151, évolutivement conservé dans les espèces possédant des cils motiles. J'ai pu montrer que CCDC151 est impliquée dans le transport dépendant de l'IFT des bras de dynéine chez les animaux et qu'elle est nécessaire à la perception sensorielle chez la drosophile. Par ailleurs, j'ai également montré que cette protéine possède des fonctions cellulaires additionnelles puisqu'elle est requise pour l'orientation correcte des plans de division cellulaire et qu'elle est impliquée dans la régulation de la taille du cil primaire chez les mammifères. Je me suis ensuite intéressée au gène LRRC48 également conservée dans les espèces possédant des cils motiles. Cette protéine est nécessaire à la motilité des flagelles de spermatozoïdes et des cils des neurones sensoriels en 9+0 et dans la réponse auditive chez la drosophile. De plus LRRC48 est indispensable au développement des vertébrés puisque son absence chez le poisson zèbre conduit à l'hydrocéphalie, des kystes rénaux et des défauts de motilité des cils. Elle est également essentielle à la biogenèse de l'oreille dans cet organisme.En conclusion, il s'agit de deux nouveaux acteurs de la ciliogenèse potentiellement impliqués dans les pathologies ciliaires chez l'Homm

Contrôle transcriptionnel de la ciliogenèse au cours du développement animal

Les cils et les flagelles sont des organelles cellulaires de structure très conservée et aux fonctions essentielles dans de nombreuses espèces, des protozoaires à l’Homme. Chez les animaux, différents types de cils coexistent et leur assemblage est dynamique au cours du développement. Chez l’Homme, toute perturbation de l’assemblage ou de la fonction des cils est responsable de nombreux syndromes désormais connus sous le terme de ciliopathies. Comprendre comment est régulé l’assemblage dynamique des cils au cours du développement et dans les différents tissus représente une question importante en biologie. Il apparaît que deux familles de facteurs de transcription, FOXJ1 (Forkhead Box J1) et RFX (Regulatory Factor X) sont d’importants acteurs du contrôle de l’expression des gènes ciliaires. FOXJ1 est essentiel à l’assemblage des cils motiles chez les vertébrés en régulant les gènes impliqués dans la motilité ciliaire ou dans le transport apical du corps basal, alors que les protéines RFX sont nécessaires à l’assemblage des cils primaires et motiles et régulent notamment les protéines du transport intraflagellaire. Récemment, de nouveaux acteurs jouant des rôles plus spécifiques dans la biogenèse des cils ont été décrits. Tous ces acteurs sont sujets à une régulation complexe permettant d’assurer le contrôle spécifique et dynamique de la ciliogenèse chez les métazoaires

Contrôle transcriptionnel des gènes ciliaires

Les cils, présents à la surface des cellules de nombreux eucaryotes, partagent une architecture commune qui peut se décliner en de nombreuses variations au sein d’une même espèce. La genèse des cils et la mise en place de leur diversité requièrent l’implication de gènes spécifiques. Ce contrôle est assuré par au moins deux classes de facteurs de transcription : les facteurs RFX (regulatory factor X), essentiels à l’assemblage de la plupart des cils, et les facteurs FOXJ1 (forkhead box J1), régulateurs clés de la croissance des cils mobiles. Ces facteurs ont des cibles distinctes et communes, et peuvent coopérer pour permettre la formation des cils. En collaborant avec d’autres facteurs de transcription spécifiques de différents types cellulaires, ils participent également à la spécialisation des cils. L’identification des gènes cibles des facteurs RFX et FOXJ1 est apparue comme une stratégie efficace pour identifier de nouveaux gènes ciliaires potentiellement impliqués dans les ciliopathies

Somatic mutations alter the differentiation outcomes of iPSC-derived neurons

The use of induced pluripotent stem cells (iPSC) as models for development and human disease has enabled the study of otherwise inaccessible tissues. A remaining challenge in developing reliable models is our limited understanding of the factors driving irregular differentiation of iPSCs, particularly the impact of acquired somatic mutations. We leveraged data from a pooled dopaminergic neuron differentiation experiment of 238 iPSC lines profiled with single-cell RNA and whole-exome sequencing to study how somatic mutations affect differentiation outcomes. We found that deleterious somatic mutations in key developmental genes, notably the BCOR gene, are strongly associated with failure in dopaminergic neuron differentiation and a larger proliferation rate in culture. We further identified broad differences in cell type composition between incorrectly and successfully differentiating lines, as well as significant changes in gene expression contributing to the inhibition of neurogenesis. Our work calls for caution in interpreting differentiation-related phenotypes in disease-modeling experiments.Peer reviewe

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

Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in Drosophila

International audienceCilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. Here, we show that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. We identify cell type specific roles of this functional module in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization

Quantitative mass spectrometry for human melanocortin peptides in vitro and in vivo suggests prominent roles for β-MSH and desacetyl α-MSH in energy homeostasis.

OBJECTIVE: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. METHODS: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. RESULTS: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of β-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin. CONCLUSIONS: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and β-MSH are likely to be the predominant physiological products acting on melanocortin receptors

Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation.

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.Open Target