Ciliogenesis and cerebrospinal fluid flow in the developing Xenopus brain are regulated by foxj1

Background: Circulation of cerebrospinal fluid (CSF) through the ventricular system is driven by motile cilia on ependymal cells of the brain. Disturbed ciliary motility induces the formation of hydrocephalus, a pathological accumulation of CSF resulting in ventricle dilatation and increased intracranial pressure. The mechanism by which loss of motile cilia causes hydrocephalus has not been elucidated. The aim of this study was: (1) to provide a detailed account of the development of ciliation in the brain of the African clawed frog Xenopus laevis; and (2) to analyze the relevance of ependymal cilia motility for CSF circulation and brain ventricle morphogenesis in Xenopus. Methods: Gene expression analysis of foxj1, the bona fide marker for motile cilia, was used to identify potentially ciliated regions in the developing central nervous system (CNS) of the tadpole. Scanning electron microscopy (SEM) was used to reveal the distribution of mono- and multiciliated cells during successive stages of brain morphogenesis, which was functionally assessed by bead injection and video microscopy of ventricular CSF flow. An antisense morpholino oligonucleotide (MO)-mediated gene knock-down that targeted foxj1 in the CNS was applied to assess the role of motile cilia in the ventricles. Results: RNA transcripts of foxj1 in the CNS were found from neurula stages onwards. Following neural tube closure, foxj1 expression was seen in distinct ventricular regions such as the zona limitans intrathalamica (ZLI), subcommissural organ (SCO), floor plate, choroid plexus (CP), and rhombomere boundaries. In all areas, expression of foxj1 preceded the outgrowth of monocilia and the subsequent switch to multiciliated ependymal cells. Cilia were absent in foxj1 morphants, causing impaired CSF flow and fourth ventricle hydrocephalus in tadpole-stage embryos. Conclusions: Motile ependymal cilia are important organelles in the Xenopus CNS, as they are essential for the circulation of CSF and maintenance of homeostatic fluid pressure. The Xenopus CNS ventricles might serve as a novel model system for the analysis of human ciliary genes whose deficiency cause hydrocephalus

ATP4 and ciliation in the neuroectoderm and endoderm of Xenopus embryos and tadpoles

AbstractDuring gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/β-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516–527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/β-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/β-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development

Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated

Role of hmmr during neurulation and brain development in the African clawed frog Xenopus laevis

Die Cerebrospinalflüssigkeit (CSF) füllt das komplette Ventrikelsystem des Hirns, den Spinalkanal und den subarachnoidalen Raum aus. Die CSF dient der mechanischen Pufferung des Hirns, transportiert Signalmoleküle und eliminiert Abfallprodukte des Ependyms. Der Plexus choroideus (PC) sekretiert die CSF welche mittels motiler Cilien innerhalb der Ventrikelräume transportiert werden. Eine übermäßige Bildung, verminderter Transport sowie verringerte Absorption der CSF können zu Hydrocephalus führen, einer pathologischen Erweiterung der Hirnventrikel. Durch Mutationen in Mensch und Maus ist bekannt, dass dysfunktionale und immotile Cilien ebenfalls zum Krankheitsbild des Hydrocephalus führen. Auf welche Weise die Beeinträchtigung von Cilienmotilität zur Ausbildung eines Hydrocpehalus führt ist bislang nicht geklärt. In der vorliegenden Arbeit wurde der embryologische Modellorganismus Xenopus laevis verwendet, um die Entstehung von Cilienmotilitäts-basiertem Hydrocephalus zu analysieren. Die Entwicklung von Cilien im Hirn von Xenopus laevis wurde bis hin zur Metamorphose beschrieben. Dabei korrelierte die Genexpression von foxj1, dem übergeordneten Regulator der Biogenese motiler Cilien, mit der Ausbildung elongierter Monocilien und dem Übergang zu Multicilien-tragenden, ependymalen Zellen. Die Cilien foxj1-positiver Zellen waren motil und erzeugten eine gerichtete CSF-Strömung. Der Funktionsverlust von foxj1 beeinträchtigte und ablatierte motile Cilien und erzeugte eine hydrocephalische Aufweitung des vierten Ventrikels. Dabei korrelierte die Entstehung von Hydrocephalus mit einer Verlangsamung der ciliengetriebenen CSF-Fließgeschwindigkeit auf unter 300 µm/s. Für Atemwegscilien ist eine Regulation des Cilienschlags durch HMMR beschrieben, wobei ein Funktionsverlust von HMMR die Cilienschlagfrequenz verlangsamt. Passend dazu führte der Funktionsverlust von hmmr in Xenopus laevis zu verlangsamter CSF-Strömung und resultierte in einem Hydrocephalus des vierten Ventrikels. Dies legt nahe, dass insbesondere im vierten Ventrikel eine CSF-Fließgeschwindigkeit von über 300 μm/s nötig ist, um einen homöostatischen Flüssigkeitsdruck im gesamten Ventrikelsystem aufrechtzuerhalten. Darüber hinaus induzierte der Funktionsverlust sowohl von foxj1 als auch von hmmr Fehlbildungen im Bereich der dorsalen Mittellinie des Vorder- und Mittelhirns. Dies betraf vor allem den PC sowie das subkommissurale Organ und somit cilienbesetzte Strukturen, welche bereits mit der Entstehung von Hydrocephalus verknüpft wurden. Die Hirndefekte nach Funktionsverlust von hmmr entsprachen dem Krankheitsbild der Holoprosencephalie (HPE). Diese kongenitale Fehlbildung wird meist durch Mutationen im Shh-Signalweg ausgelöst und geht mit Hydrocephalus einher. Überraschenderweise war HPE nach Funktionsverlust von hmmr unabhängig vom Shh-Signalweg. Vielmehr war die Entwicklung des Vorderhirns gestört, da hmmr für Mikrotubuli-vermittelte Zelladhäsion während der morphogenetischen Bewegungen der Neurulation notwendig war. Diese Arbeit zeigte zum ersten Mal, dass die CSF in Xenopus laevis durch motile Cilien transportiert wird und bestätigte, dass fehlende und dysfunktionale, motile Cilien zu kongenitalem Hydrocephalus führen. Neu ist, dass motile Cilien eine Rolle für die Morphogenese des Vorder- und Mittelhirns spielen. Die Enstehung eines Hydrocephalus im Zusammenhang mit Vorderhirndefekten in foxj1- und hmmr-Morphanten legt nahe, dass ein cilienabhängiger Hydrocephalus durch die Fehlbildung dorsaler Mittellinienstrukturen entstehen kann. Die vorliegende Arbeit hat somit die Grundlage geschaffen, um Xenopus laevis als Modellsystem für die Entstehung von Hydrocephalus bei ciliärer Dyskinesie und Vorderhirndefekten zu etablieren.The cerebrospinal fluid (CSF) fills the entire ventricular system of the brain, the spinal cavity and the subarachnoid space. CSF mechanically buffers the brain, transports signaling molecules and eliminates waste products. It is produced by the choroid plexus (CP) and transported throughout the ventricular system via motile cilia. Excessive production, diminished transport or reduced absorption of CSF lead to hydrocephalus, a pathological dilatation of the brain ventricles. Mutations in humans and mice showed that dysfunctional and immotile cilia also induce hydrocephalus. The underlying mechanism through which disturbed ciliary motility leads to formation of hydrocephalus is not resolved. In the present thesis the model organism Xenopus laevis was used to analyze the occurrence of hydrocephalus upon on ciliary dysmotility. Biogenesis of motile cilia was described in the Xenopus laevis brain up to metamorphosis. Gene expression of foxj1, the superior regulator of the biogenesis of motile cilia, correlated with development of elongated monocilia and the switch to multiciliated ependymal cells. Cilia on foxj1-positive cells were motile and produced a directional flow of CSF. foxj1 loss-of-function led to impaired or absent motile cilia and resulted in hydrocephalus. The development of the hydrocephalic dilatation correlated with reduced velocity of the cilia-driven CSF-flow below 300 µm/s. In cilia of the airway epithelium regulation of ciliary beat frequency via HMMR has been described with HMMR loss-of-function resulting in reduced ciliary beat frequency. In line with these results, hmmr loss-of-function in Xenopus laevis resulted in reduced velocity of CSF-flow and hydrocephalus. This suggests that especially in the fourth ventricle CSF-flow velocities above 300 µm/s are necessary to maintain a homeostatic fluid pressure in the entire ventricular system. The loss-of-function of foxj1 as well as hmmr further led to severe malformations in the dorsal midline of the brain, especially of the CP and the subcommissural organ. These ciliated structures have already been connected to development of hydrocephalus. Brain defects after loss-of-function of hmmr reflected the human disorder of holoprosencephaly (HPE) which often results from mutations in the Shh-signaling pathway and leads to hydrocephalus. Interestingly after hmmr loss-of-function induced HPE was independent of the Shh-signaling pathway. Forebrain development was disturbed because hmmr was necessary for microtubule-mediated cell adhesion during the morphogenetic movements of neurulation. This study shows for the first time, that CSF in Xenopus laevis is transported via motile cilia and confirmes that dysfunction or absent motile cilia lead to congenital hydrocephalus. Furthermore a novel role for motile cilia during fore- and midbrain morphogenesis was demonstrated. Development of hydrocephalus together with forebrain defects in foxj1 and hmmr morphants implies that cilia-dependent hydrocephalus can result from malformed dorsal midline structures. This study thus provides a basis to establish Xenopus laevis as a model organism to study the development of hydrocephalus caused by primary cilia dyskinesia and by forebrain defects

RESEARCH Open Access

Ciliogenesis and cerebrospinal fluid flow in the developing Xenopus brain are regulated by foxj

ATP4a is required for development and function of the Xenopus mucociliary epidermis – a potential model to study proton pump inhibitor-associated pneumonia

Proton pump inhibitors (PPIs), which target gastric H(+)/K(+)ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/β-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways

Calponin 2 Acts As an Effector of Noncanonical Wnt-Mediated Cell Polarization during Neural Crest Cell Migration

Neural crest cells (NCCs) migrate throughout the embryo to differentiate into cell types of all germ layers. Initial directed NCC emigration relies on planar cell polarity (PCP), which through the activity of the small GTPases RhoA and Rac governs the actin-driven formation of polarized cell protrusions. We found that the actin binding protein calponin 2 (Cnn2) was expressed in protrusions at the leading edge of migratory NCCs in chicks and frogs. Cnn2 knockdown resulted in NCC migration defects in frogs and chicks and randomized outgrowth of cell protrusions in NCC explants. Morphant cells showed central stress fibers at the expense of the peripheral actin network. Cnn2 acted downstream of Wnt/PCP, as migration defects induced by dominant-negative Wnt11 or inhibition of RhoA function were rescued by Cnn2 knockdown. These results suggest that Cnn2 modulates actin dynamics during NCC migration as an effector of noncanonical Wnt/PCP signaling

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.publishe