Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

p53/p21 pathway activation contributes to the ependymal fate decision downstream of GemC1.

Multiciliated ependymal cells and adult neural stem cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here, we show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, is induced, without affecting cell survival. Genotoxic stress is not sufficient by itself to induce ependymal cell differentiation, although the absence of p53 or p21 in progenitors hinders differentiation by maintaining cell division. Activation of the p53-p21 pathway downstream of GemC1 leads to cell-cycle slowdown/arrest, which permits timely onset of ependymal cell differentiation in progenitor cells.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

Massive centriole production can occur in the absence of deuterosomes in multiciliated cells.

Multiciliated cells (MCCs) amplify large numbers of centrioles that convert into basal bodies, which are required for producing multiple motile cilia. Most centrioles amplified by MCCs grow on the surface of organelles called deuterosomes, whereas a smaller number grow through the centriolar pathway in association with the two parent centrioles. Here, we show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. Therefore, deuterosomes may have evolved to relieve, rather than supplement, the centriolar pathway during multiciliogenesis. Remarkably, MCCs lacking parent centrioles and deuterosomes also amplify the appropriate number of centrioles inside a cloud of pericentriolar and fibrogranular material. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize.

Dynamics of centriole amplification in centrosome-depleted brain multiciliated progenitors.

Reproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack the centrosome duplication pathway to form hundreds of centrioles and nucleate motile cilia. In these cells, the large majority of procentrioles are formed associated with partially characterized organelles called deuterosomes. We recently challenged the paradigm that deuterosomes and procentrioles are formed de novo by providing data, in brain MCC, suggesting that they are nucleated from the pre-existing centrosomal younger centriole. However, the origin of deuterosomes and procentrioles is still under debate. Here, we further question centrosome importance for deuterosome and centriole amplification. First, we provide additional data confirming that centriole amplification occurs sequentially from the centrosomal region, and that the first procentriole-loaded deuterosomes are associated with the daughter centriole or in the centrosomal centriole vicinity. Then, to further test the requirement of the centrosome in deuterosome and centriole formation, we depleted centrosomal centrioles using a Plk4 inhibitor. We reveal unexpected limited consequences in deuterosome/centriole number in absence of centrosomal centrioles. Notably, in absence of the daughter centriole only, deuterosomes are not seen associated with the mother centriole. In absence of both centrosomal centrioles, procentrioles are still amplified sequentially and with no apparent structural defects. They seem to arise from a focal region, characterized by microtubule convergence and pericentriolar material (PCM) assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed.

Adult Neural Stem Cells and Multiciliated Ependymal Cells Share a Common Lineage Regulated by the Geminin Family Members.

Adult neural stem cells and multiciliated ependymal cells are glial cells essential for neurological functions. Together, they make up the adult neurogenic niche. Using both high-throughput clonal analysis and single-cell resolution of progenitor division patterns and fate, we show that these two components of the neurogenic niche are lineally related: adult neural stem cells are sister cells to ependymal cells, whereas most ependymal cells arise from the terminal symmetric divisions of the lineage. Unexpectedly, we found that the antagonist regulators of DNA replication, GemC1 and Geminin, can tune the proportion of neural stem cells and ependymal cells. Our findings reveal the controlled dynamic of the neurogenic niche ontogeny and identify the Geminin family members as key regulators of the initial pool of adult neural stem cells.

Motile ciliogenesis and the mitotic prism.

Motile cilia of epithelial multiciliated cells transport vital fluids along organ lumens to promote essential respiratory, reproductive and brain functions. Progenitors of multiciliated cells undergo massive and coordinated organelle remodelling during their differentiation for subsequent motile ciliogenesis. Defects in multiciliated cell differentiation lead to severe cilia-related diseases by perturbing cilia-based flows. Recent work designated the machinery of mitosis as the orchestrator of the orderly progression of differentiation associated with multiple motile cilia formation. By examining the events leading to motile ciliogenesis with a methodological prism of mitosis, we contextualise and discuss the recent findings to broaden the spectrum of questions related to the differentiation of mammalian multiciliated cells.

Ependymal cilia beating induces an actin network to protect centrioles against shear stress.

Multiciliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability.

Calibrated mitotic oscillator drives motile ciliogenesis.

Cell division and differentiation depend on massive and rapid organelle remodeling. The mitotic oscillator, centered on the cyclin-dependent kinase 1-anaphase-promoting complex/cyclosome (CDK1-APC/C) axis, spatiotemporally coordinates this reorganization in dividing cells. Here we discovered that nondividing cells could also implement this mitotic clocklike regulatory circuit to orchestrate subcellular reorganization associated with differentiation. We probed centriole amplification in differentiating mouse-brain multiciliated cells. These postmitotic progenitors fine-tuned mitotic oscillator activity to drive the orderly progression of centriole production, maturation, and motile ciliation while avoiding the mitosis commitment threshold. Insufficient CDK1 activity hindered differentiation, whereas excessive activity accelerated differentiation yet drove postmitotic progenitors into mitosis. Thus, postmitotic cells can redeploy and calibrate the mitotic oscillator to uncouple cytoplasmic from nuclear dynamics for organelle remodeling associated with differentiation.

The development and functions of multiciliated epithelia.

Multiciliated cells are epithelial cells that are in contact with bodily fluids and are required for the proper function of major organs including the brain, the respiratory system and the reproductive tracts. Their multiple motile cilia beat unidirectionally to remove particles of external origin from their surface and/or drive cells or fluids into the lumen of the organs. Multiciliated cells in the brain are produced once, almost exclusively during embryonic development, whereas in respiratory tracts and oviducts they regenerate throughout life. In this Review, we provide a cell-to-organ overview of multiciliated cells and highlight recent studies that have greatly increased our understanding of the mechanisms driving the development and function of these cells in vertebrates. We discuss cell fate determination and differentiation of multiciliated cells, and provide a comprehensive account of their locations and functions in mammals.

Multiciliated Cells in Animals.

Many animal cells assemble single cilia involved in motile and/or sensory functions. In contrast, multiciliated cells (MCCs) assemble up to 300 motile cilia that beat in a coordinate fashion to generate a directional fluid flow. In the human airways, the brain, and the oviduct, MCCs allow mucus clearance, cerebrospinal fluid circulation, and egg transportation, respectively. Impairment of MCC function leads to chronic respiratory infections and increased risks of hydrocephalus and female infertility. MCC differentiation during development or repair involves the activation of a regulatory cascade triggered by the inhibition of Notch activity in MCC progenitors. The downstream events include the simultaneous assembly of a large number of basal bodies (BBs)-from which cilia are nucleated-in the cytoplasm of the differentiating MCCs, their migration and docking at the plasma membrane associated to an important remodeling of the actin cytoskeleton, and the assembly and polarization of motile cilia. The direction of ciliary beating is coordinated both within cells and at the tissue level by a combination of planar polarity cues affecting BB position and hydrodynamic forces that are both generated and sensed by the cilia. Herein, we review the mechanisms controlling the specification and differentiation of MCCs and BB assembly and organization at the apical surface, as well as ciliary assembly and coordination in MCCs.

Centriole continuity: out with the new, in with the old.

Centrioles are essential microtubule-based organelles, typically present in pairs, which organize cilia and centrosomes. Their mode of biogenesis is unique for a subcellular organelle since, during cell division, each pre-existing centriole guides the formation of a new one, a process that is coordinated with DNA replication. After centriole duplication, the new centrosomes migrate in opposite direction and localize at each pole of the mitotic spindle. This singular dynamics led to think that centrioles were permanent self-replicating structures coordinating cytoplasm and nuclear division. This vision then fell gradually into disuse when centrioles were shown to be capable to form de novo, in the absence of a pre-existing structure, and to be actually dispensable for cell division. However, new data, which are reviewed here, have breathed new life into the old ideas.

Ependymal cell differentiation, from monociliated to multiciliated cells.

Primary and motile cilia differ in their structure, composition, and function. In the brain, primary cilia are immotile signalling organelles present on neural stem cells and neurons. Multiple motile cilia are found on the surface of ependymal cells in all brain ventricles, where they contribute to the flow of cerebrospinal fluid. During development, monociliated ependymal progenitor cells differentiate into multiciliated ependymal cells, thus providing a simple system for studying the transition between these two stages. In this chapter, we provide protocols for immunofluorescence staining of developing ependymal cells in vivo, on whole mounts of lateral ventricle walls, and in vitro, on cultured ependymal cells. We also provide a list of markers we currently use to stain both types of cilia, including proteins at the ciliary membrane and tubulin posttranslational modifications of the axoneme.

Centriole amplification by mother and daughter centrioles differs in multiciliated cells.

The semi-conservative centrosome duplication in cycling cells gives rise to a centrosome composed of a mother and a newly formed daughter centriole. Both centrioles are regarded as equivalent in their ability to form new centrioles and their symmetric duplication is crucial for cell division homeostasis. Multiciliated cells do not use the archetypal duplication program and instead form more than a hundred centrioles that are required for the growth of motile cilia and the efficient propelling of physiological fluids. The majority of these new centrioles are thought to appear de novo, that is, independently from the centrosome, around electron-dense structures called deuterosomes. Their origin remains unknown. Using live imaging combined with correlative super-resolution light and electron microscopy, we show that all new centrioles derive from the pre-existing progenitor cell centrosome through multiple rounds of procentriole seeding. Moreover, we establish that only the daughter centrosomal centriole contributes to deuterosome formation, and thus to over ninety per cent of the final centriole population. This unexpected centriolar asymmetry grants new perspectives when studying cilia-related diseases and pathological centriole amplification observed in cycling cells and associated with microcephaly and cancer.

Analysis of human samples reveals impaired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome.

Joubert syndrome (JS) and Meckel syndrome (MKS) are pleiotropic ciliopathies characterized by severe defects of the cerebellar vermis, ranging from hypoplasia to aplasia. Interestingly, ciliary conditional mutant mice have a hypoplastic cerebellum in which the proliferation of cerebellar granule cell progenitors (GCPs) in response to Sonic hedgehog (SHH) is severely reduced. This suggests that Shh signaling defects could contribute to the vermis hypoplasia observed in the human syndromes. As existing JS/MKS mutant mouse models suggest apparently contradictory hypotheses on JS/MKS etiology, we investigated Shh signaling directly on human fetal samples. First, in an examination of human cerebellar development, we linked the rates of GCP proliferation to the different levels and localizations of active Shh signaling and showed that the GCP possessed a primary cilium with CEP290 at its base. Second, we found that the proliferation of GCPs and their response to SHH were severely impaired in the cerebellum of subjects with JS/MKS and Jeune syndrome. Finally, we showed that the defect in GCP proliferation was similar in the cerebellar vermis and hemispheres in all patients with ciliopathy analyzed, suggesting that the specific cause of vermal hypo-/aplasia precedes this defect. Our results, obtained from the analysis of human samples, show that the hemispheres and the vermis are affected in JS/MKS and provide evidence of a defective cellular mechanism in these pathologic processes.

Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II.

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia.

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia.

In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

Gene-based approaches in the study of pathological pain.

Chronic pathological pain is characterized by extensive plasticity of the systems involved in pain signal transmission and modulation and tissue remodeling in several CNS structures. These long-lasting alterations are mediated by, or associated with, changes in the production of key molecules of nociceptive processing. Gene-based approaches offer the unique possibility of using local or even cell-type specific interventions to correct the abnormal production of some of these proteins, modulate the activity of signal transduction pathways, or overproduce various therapeutic secreted proteins. We showed that certain viral-derived vectors are particularly suitable for mediating gene transfer highly preferential for instance into the primary sensory neurons or into the spinal cord glial cells that represent particularly pertinent targets in the search for new therapeutic strategies of pathological pain.