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.

Biallelic Mutations in TMTC3, Encoding a Transmembrane and TPR-Containing Protein, Lead to Cobblestone Lissencephaly.

Cobblestone lissencephaly (COB) is a severe brain malformation in which overmigration of neurons and glial cells into the arachnoid space results in the formation of cortical dysplasia. COB occurs in a wide range of genetic disorders known as dystroglycanopathies, which are congenital muscular dystrophies associated with brain and eye anomalies and range from Walker-Warburg syndrome to Fukuyama congenital muscular dystrophy. Each of these conditions has been associated with alpha-dystroglycan defects or with mutations in genes encoding basement membrane components, which are known to interact with alpha-dystroglycan. Our screening of a cohort of 25 families with recessive forms of COB identified six families affected by biallelic mutations in TMTC3 (encoding transmembrane and tetratricopeptide repeat containing 3), a gene without obvious functional connections to alpha-dystroglycan. Most affected individuals showed brainstem and cerebellum hypoplasia, as well as ventriculomegaly. However, the minority of the affected individuals had eye defects or elevated muscle creatine phosphokinase, separating the TMTC3 COB phenotype from typical congenital muscular dystrophies. Our data suggest that loss of TMTC3 causes COB with minimal eye or muscle involvement.

Biallelic truncating mutations in FMN2, encoding the actin-regulatory protein Formin 2, cause nonsyndromic autosomal-recessive intellectual disability.

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.

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

Cilia 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.

[Transcriptional control of ciliary genes]. / Contrôle transcriptionnel des gènes ciliaires.

Cilia are found in many eukaryotic species and share a common microtubule architecture that can nonetheless show very diverse features within one animal. The genesis of cilia and their diversity require the expression of different specific genes. At least two classes of transcription factors are involved in ciliogenesis: the RFX family, essential for the assembly of most cilia and the FOXJ1 transcription factors that are key regulators of motile cilia assembly. These two different families of transcription factors have both specific and common target genes and they can also cooperate for the formation of cilia. In collaboration with cell type specific factors, they also contribute to the specialisation of cilia. As a consequence, the identification of RFX and FOXJ1 target genes has emerged as an efficient strategy to identify novel ciliary genes, and in particular genes potentially implicated in 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.

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.

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

Cilia 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 ß-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 ß-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 ß-MSH are likely to be the predominant physiological products acting on melanocortin receptors.

[Transcriptional control of ciliogenesis in animal development]. / Contrôle transcriptionnel de la ciliogenèse au cours du développement animal.

Cilia and flagella are eukaryotic organelles with a conserved structure and function from unicellular organisms to human. In animals, different types of cilia can be found and cilia assembly during development is a highly dynamic process. Ciliary defects in human lead to a wide spectrum of diseases called ciliopathies. Understanding the molecular mechanisms that govern dynamic cilia assembly during development and in different tissues in metazoans is an important biological challenge. The FOXJ1 (Forkhead Box J1) and RFX (Regulatory Factor X) family of transcription factors have been shown to be important factors in ciliogenesis control. FOXJ1 proteins are required for motile ciliogenesis in vertebrates. By contrast, RFX proteins are essential to assemble both primary and motile cilia through the regulation of specific sets of genes such as those encoding intraflagellar transport components. Recently, new actors with more specific roles in cilia biogenesis and physiology have also been discovered. All these factors are subject to complex regulation, allowing for the dynamic and specific regulation of ciliogenesis in metazoans.