Emerging principles of primary cilia dynamics in controlling tissue organization and function.

Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context-dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell- and tissue-specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.

The forkhead transcription factor Foxj1 controls vertebrate olfactory cilia biogenesis and sensory neuron differentiation.

In vertebrates, olfactory receptors localize on multiple cilia elaborated on dendritic knobs of olfactory sensory neurons (OSNs). Although olfactory cilia dysfunction can cause anosmia, how their differentiation is programmed at the transcriptional level has remained largely unexplored. We discovered in zebrafish and mice that Foxj1, a forkhead domain-containing transcription factor traditionally linked with motile cilia biogenesis, is expressed in OSNs and required for olfactory epithelium (OE) formation. In keeping with the immotile nature of olfactory cilia, we observed that ciliary motility genes are repressed in zebrafish, mouse, and human OSNs. Strikingly, we also found that besides ciliogenesis, Foxj1 controls the differentiation of the OSNs themselves by regulating their cell type-specific gene expression, such as that of olfactory marker protein (omp) involved in odor-evoked signal transduction. In line with this, response to bile acids, odors detected by OMP-positive OSNs, was significantly diminished in foxj1 mutant zebrafish. Taken together, our findings establish how the canonical Foxj1-mediated motile ciliogenic transcriptional program has been repurposed for the biogenesis of immotile olfactory cilia, as well as for the development of the OSNs.

Expression of taste sentinels, T1R, T2R, and PLCß2, on the passageway for olfactory signals in zebrafish.

The senses of taste and smell detect overlapping sets of chemical compounds in fish, e.g. amino acids are detected by both senses. However, so far taste and smell organs appeared morphologically to be very distinct, with a specialized olfactory epithelium for detection of odors and taste buds located in the oral cavity and lip for detection of tastants. Here, we report dense clusters of cells expressing T1R and T2R receptors as well as their signal transduction molecule PLCß2 in nostrils of zebrafish, i.e. on the entrance funnel through which odor molecules must pass to be detected by olfactory sensory neurons. Quantitative evaluation shows the density of these chemosensory cells in the nostrils to be as high or higher than that in the established taste organs oral cavity and lower lip. Hydrodynamic flow is maximal at the nostril rim enabling high throughput chemosensation in this organ. Taken together, our results suggest a sentinel function for these chemosensory cells in the nostril.

Loss of glutamate transporter eaat2a leads to aberrant neuronal excitability, recurrent epileptic seizures, and basal hypoactivity.

Astroglial excitatory amino acid transporter 2 (EAAT2, GLT-1, and SLC1A2) regulates the duration and extent of neuronal excitation by removing glutamate from the synaptic cleft. Hence, an impairment in EAAT2 function could lead to an imbalanced brain network excitability. Here, we investigated the functional alterations of neuronal and astroglial networks associated with the loss of function in the astroglia predominant eaat2a gene in zebrafish. We observed that eaat2a-/- mutant zebrafish larvae display recurrent spontaneous and light-induced seizures in neurons and astroglia, which coincide with an abrupt increase in extracellular glutamate levels. In stark contrast to this hyperexcitability, basal neuronal and astroglial activity was surprisingly reduced in eaat2a-/- mutant animals, which manifested in decreased overall locomotion. Our results reveal an essential and mechanistic contribution of EAAT2a in balancing brain excitability, and its direct link to epileptic seizures.

Elevated photic response is followed by a rapid decay and depressed state in ictogenic networks.

OBJECTIVE: The switch between nonseizure and seizure states involves profound alterations in network excitability and synchrony. In this study, we aimed to identify and compare features of neural excitability and dynamics across multiple zebrafish seizure and epilepsy models. METHODS: Inspired by video-electroencephalographic recordings in patients, we developed a framework to study spontaneous and photically evoked neural and locomotor activity in zebrafish larvae, by combining high-throughput behavioral tracking and whole-brain in vivo two-photon calcium imaging. RESULTS: Our setup allowed us to dissect behavioral and physiological features that are divergent or convergent across multiple models. We observed that spontaneous locomotor and neural activity exhibit great diversity across models. Nonetheless, during photic stimulation, hyperexcitability and rapid response dynamics were well conserved across multiple models, highlighting the reliability of photically evoked activity for high-throughput assays. Intriguingly, in several models, we observed that the initial elevated photic response is often followed by rapid decay of neural activity and a prominent depressed state. Elevated photic response and following depressed state in seizure-prone networks are significantly reduced by the antiseizure medication valproic acid. Finally, rapid decay and depression of neural activity following photic stimulation temporally overlap with slow recruitment of astroglial calcium signals that are enhanced in seizure-prone networks. SIGNIFICANCE: We argue that fast decay of neural activity and depressed states following photic response are likely due to homeostatic mechanisms triggered by excessive neural activity. An improved understanding of the interplay between elevated and depressed excitability states might suggest tailored epilepsy therapies.

CiliaQ: a simple, open-source software for automated quantification of ciliary morphology and fluorescence in 2D, 3D, and 4D images.

Cilia are hair-like membrane protrusions that emanate from the surface of most vertebrate cells and are classified into motile and primary cilia. Motile cilia move fluid flow or propel cells, while also fulfill sensory functions. Primary cilia are immotile and act as a cellular antenna, translating environmental cues into cellular responses. Ciliary dysfunction leads to severe diseases, commonly termed ciliopathies. The molecular details underlying ciliopathies and ciliary function are, however, not well understood. Since cilia are small subcellular compartments, imaging-based approaches have been used to study them. However, tools to comprehensively analyze images are lacking. Automatic analysis approaches require commercial software and are limited to 2D analysis and only a few parameters. The widely used manual analysis approaches are time consuming, user-biased, and difficult to compare. Here, we present CiliaQ, a package of open-source, freely available, and easy-to-use ImageJ plugins. CiliaQ allows high-throughput analysis of 2D and 3D, static or time-lapse images from fluorescence microscopy of cilia in cell culture or tissues, and outputs a comprehensive list of parameters for ciliary morphology, length, bending, orientation, and fluorescence intensity, making it broadly applicable. We envision CiliaQ as a resource and platform for reproducible and comprehensive analysis of ciliary function in health and disease.

Radial glia in the zebrafish brain: Functional, structural, and physiological comparison with the mammalian glia.

The neuroscience community has witnessed a tremendous expansion of glia research. Glial cells are now on center stage with leading roles in the development, maturation, and physiology of brain circuits. Over the course of evolution, glia have highly diversified and include the radial glia, astroglia or astrocytes, microglia, oligodendrocytes, and ependymal cells, each having dedicated functions in the brain. The zebrafish, a small teleost fish, is no exception to this and recent evidences point to evolutionarily conserved roles for glia in the development and physiology of its nervous system. Due to its small size, transparency, and genetic amenability, the zebrafish has become an increasingly prominent animal model for brain research. It has enabled the study of neural circuits from individual cells to entire brains, with a precision unmatched in other vertebrate models. Moreover, its high neurogenic and regenerative potential has attracted a lot of attention from the research community focusing on neural stem cells and neurodegenerative diseases. Hence, studies using zebrafish have the potential to provide fundamental insights about brain development and function, and also elucidate neural and molecular mechanisms of neurological diseases. We will discuss here recent discoveries on the diverse roles of radial glia and astroglia in neurogenesis, in modulating neuronal activity and in regulating brain homeostasis at the brain barriers. By comparing insights made in various animal models, particularly mammals and zebrafish, our goal is to highlight the similarities and differences in glia biology among species, which could set new paradigms relevant to humans.

Noncanonical translation via deadenylated 3' UTRs maintains primordial germ cells.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.

A fast growing spectrum of biological functions of γ-secretase in development and disease.

γ-secretase, which assembles as a tetrameric complex, is an aspartyl protease that proteolytically cleaves substrate proteins within their membrane-spanning domain; a process also known as regulated intramembrane proteolysis (RIP). RIP regulates signaling pathways by abrogating or releasing signaling molecules. Since the discovery, already >15 years ago, of its catalytic component, presenilin, and even much earlier with the identification of amyloid precursor protein as its first substrate, γ-secretase has been commonly associated with Alzheimer's disease. However, starting with Notch and thereafter a continuously increasing number of novel substrates, γ-secretase is becoming linked to an equally broader range of biological processes. This review presents an updated overview of the current knowledge on the diverse molecular mechanisms and signaling pathways controlled by γ-secretase, with a focus on organ development, homeostasis and dysfunction. This article is part of a Special Issue entitled: Intramembrane Proteases.

The neuronal cilium - a highly diverse and dynamic organelle involved in sensory detection and neuromodulation.

Cilia are fascinating organelles that act as cellular antennae, sensing the cellular environment. Cilia gained significant attention in the late 1990s after their dysfunction was linked to genetic diseases known as ciliopathies. Since then, several breakthrough discoveries have uncovered the mechanisms underlying cilia biogenesis and function. Like most cells in the animal kingdom, neurons also harbor cilia, which are enriched in neuromodulatory receptors. Yet, how neuronal cilia modulate neuronal physiology and animal behavior remains poorly understood. By comparing ciliary biology between the sensory and central nervous systems (CNS), we provide new perspectives on the functions of cilia in brain physiology.

Ciliogenesis defects after neurulation impact brain development and neuronal activity in larval zebrafish.

Cilia are slender, hair-like structures extending from cell surfaces and playing essential roles in diverse physiological processes. Within the nervous system, primary cilia contribute to signaling and sensory perception, while motile cilia facilitate cerebrospinal fluid flow. Here, we investigated the impact of ciliary loss on neural circuit development using a zebrafish line displaying ciliogenesis defects. We found that cilia defects after neurulation affect neurogenesis and brain morphology, especially in the cerebellum, and lead to altered gene expression profiles. Using whole brain calcium imaging, we measured reduced light-evoked and spontaneous neuronal activity in all brain regions. By shedding light on the intricate role of cilia in neural circuit formation and function in the zebrafish, our work highlights their evolutionary conserved role in the brain and sets the stage for future analysis of ciliopathy models.

The evolutionarily conserved choroid plexus contributes to the homeostasis of brain ventricles in zebrafish.

The choroid plexus (ChP) produces cerebrospinal fluid (CSF). It also contributes to brain development and serves as the CSF-blood barrier. Prior studies have identified transporters on the epithelial cells that transport water and ions from the blood vasculature to the ventricles and tight junctions involved in the CSF-blood barrier. Yet, how the ChP epithelial cells control brain physiology remains unresolved. We use zebrafish to provide insights into the physiological roles of the ChP. Upon histological and transcriptomic analyses, we identify that the zebrafish ChP is conserved with mammals and expresses transporters involved in CSF secretion. Next, we show that the ChP epithelial cells secrete proteins into CSF. By ablating the ChP epithelial cells, we identify a reduction of the ventricular sizes without alterations of the CSF-blood barrier. Altogether, our findings reveal that the zebrafish ChP is conserved and contributes to the size and homeostasis of the brain ventricles.

Boosting NKCC1 in the choroid plexus: From CSF clearance to a potential therapy for posthemorrhagic hydrocephalus.

In this issue of Neuron, Sadegh et al.1 identify a novel potential therapeutical target for posthemorrhagic hydrocephalus (PHH). The authors identified that overexpression of Na-K-2Cl cotransporter-1 (NKCC1) in the choroid plexus relieves ventriculomegaly and enhances cerebrospinal fluid (CSF) clearance in improved PHH mouse models.

Methods to study motile ciliated cell types in the zebrafish brain.

Cilia are well conserved hair-like structures that have diverse sensory and motile functions. In the brain, motile ciliated cells, known as ependymal cells, line the cerebrospinal fluid (CSF) filled ventricles, where their beating contribute to fluid movement. Ependymal cells have gathered increasing interest since they are associated with hydrocephalus, a neurological condition with ventricular enlargement. In this article, we highlight methods to identify and characterize motile ciliated ependymal lineage in the brain of zebrafish using histological staining and transgenic reporter lines.

Novel analytical tools reveal that local synchronization of cilia coincides with tissue-scale metachronal waves in zebrafish multiciliated epithelia.

Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right noses, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, i.e., cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

Computational Modeling of Motile Cilia-Driven Cerebrospinal Flow in the Brain Ventricles of Zebrafish Embryo.

Motile cilia are hair-like microscopic structures which generate directional flow to provide fluid transport in various biological processes. Ciliary beating is one of the sources of cerebrospinal flow (CSF) in brain ventricles. In this study, we investigated how the tilt angle, quantity, and phase relationship of cilia affect CSF flow patterns in the brain ventricles of zebrafish embryos. For this purpose, two-dimensional computational fluid dynamics (CFD) simulations are performed to determine the flow fields generated by the motile cilia. The cilia are modeled as thin membranes with prescribed motions. The cilia motions were obtained from a two-day post-fertilization zebrafish embryo previously imaged via light sheet fluorescence microscopy. We observed that the cilium angle significantly alters the generated flow velocity and mass flow rates. As the cilium angle gets closer to the wall, higher flow velocities are observed. Phase difference between two adjacent beating cilia also affects the flow field as the cilia with no phase difference produce significantly lower mass flow rates. In conclusion, our simulations revealed that the most efficient method for cilia-driven fluid transport relies on the alignment of multiple cilia beating with a phase difference, which is also observed in vivo in the developing zebrafish brain.

Measurement of ciliary beating and fluid flow in the zebrafish adult telencephalon.

Motile cilia are hair-like structures that move and propel fluid, playing important roles in the physiology of organs. Here, we present a protocol to visualize and measure ciliary beating and cerebrospinal fluid (CSF) flow in the telencephalon of an adult zebrafish brain explant. We describe the preparation of brain explants, the recording of ciliary beating and CSF flow, and data analysis using ImageJ and MATLAB. These imaging and analysis techniques can be directly translated to other ciliated systems. For complete details on the use and execution of this protocol, please refer to D'Gama et al. (2021).

Past, present and future of zebrafish in epilepsy research.

Animal models contribute greatly to our understanding of brain development and function as well as its dysfunction in neurological diseases. Epilepsy research is a very good example of how animal models can provide us with a mechanistic understanding of the genes, molecules, and pathophysiological processes involved in disease. Over the course of the last two decades, zebrafish came in as a new player in epilepsy research, with an expanding number of laboratories using this animal to understand epilepsy and to discover new strategies for preventing seizures. Yet, zebrafish as a model offers a lot more for epilepsy research. In this viewpoint, we aim to highlight some key contributions of zebrafish to epilepsy research, and we want to emphasize the great untapped potential of this animal model for expanding these contributions. We hope that our suggestions will trigger further discussions between clinicians and researchers with a common goal to understand and cure epilepsy.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

Development: How the Reissner Fiber Keeps Our Back Straight.

Two new studies elegantly identify a missing link between idiopathic scoliosis and the Reissner fiber.