ATP4 and Wnt-signaling are required for ciliogenesis and left-right axis development of Xenopus

The vertebrate body plan displays left-right (LR) asymmetries of organ placement superimposed on an overt bilaterally symmetrical organization. Symmetry is broken during embryogenesis, and asymmetric gene expression precedes asymmetric organ morphogenesis. The proton/potassium pump ATP4 was shown to play a role in LR-development of the frog Xenopus laevis as well as in other deuterostomes. Two opposing models of symmetry-breakage were proposed, the ?ion-flux? and the ?leftward flow? model. The former proposed that symmetry was broken by LR-asymmetric expression of the a-subunit of ATP4 during cleavage stages. The latter claimed a cilia-based leftward flow at the gastrocoel roof plate (GRP) to take center stage during neurulation, i.e. a day later in development. In the present thesis work, the role of ATP4a in symmetry-breakage was re-addressed and evidence for symmetrical expression and function of ATP4a was gathered. ATP4a was shown to be required for two Wnt-signaling dependent steps during the setup of cilia driven leftward flow at the GRP: (1) Wnt/b-catenin (b-cat) dependent expression of Foxj1 during gastrulation, and (2) Wnt/planar cell polarity (PCP) dependent posterior localization of motile cilia during neurulation. These data challenge the ?ion-flux? hypothesis and argue for a conserved ATP4- and cilia-dependent symmetry-breakage mechanism throughout the vertebrates. Furthermore, the function of Wnt-signaling components was analyzed in the context of GRP-formation: The receptor Frizzled 8 (Fz8) and b-cat were required for Foxj1 expression during gastrulation. Morphogenesis of the GRP, posterior polarization of motile cilia and expression of Xnr1 and Coco in somitic cells were all required for LR-development. Loss of non-canonical Xwnt11b-signaling perturbed these process, suggesting that non-canonical Wnt-signaling branches, in addition to Wnt/PCP, were relevant for LR-development. ATP4-mediated Wnt-signaling was also required for Foxj1 expression and motile cilia in other epithelia during Xenopus development, i.e. the skin, floor plate and the ependymal cell layer. In the floor plate b-cat was required for Foxj1 expression downstream of Hedgehog-signaling. In the skin mucociliary epithelium ATP4a and Wnt/b-cat were required downstream of Notch/Delta-mediated cell-type specification of multiciliated cells. This was also true for a new cell type of serotonergic cells described here, which was characterized morphologically, by analysis of gene expression and response to manipulations of Wnt- and Notch/Delta-signaling. In summary, the data presented in this thesis suggest a conserved function of ATP4a and Wnt-signaling in vertebrate symmetry-breakage and Foxj1-dependent ciliogenesis in Xenopus.Wirbeltiere weisen Links-Rechts-(LR-)Asymmetrien in der Positionierung ihrer inneren Organe auf, welche von dem im Allgemeinen bilateral-symmetrischen Körperbauplan überlagert werden. Die bilaterale Symmetrie wird während der Embryonalentwicklung gebrochen, dabei geht die asymmetrische Aktivität von Genen der asymmetrischen Organmorphogenese voraus. Der Protonen/Kalium-Pumpe ATP4 wurde eine Rolle während der LR-Entwicklung von Xenopus laevis und weiteren Deuterostomiern zugesprochen. Zum Ablauf des Symmetriebruchs wurden jedoch zwei gegensätzliche Modelle vorgeschlagen: das ?Ionen-Fluss?- und das ?Flüssigkeitsstrom?-Modell. Während das erste Modell impliziert, dass eine LR-asymmetrische Verteilung der a-Untereinheit von ATP4 in Furchungsstadien zum Symmetriebruch führt, schlägt das zweite Modell vor, dass ein cilienabhängiger, linksgerichteter Flüssigkeitsstrom über Zellen der Archenteron-Dachplatte (GRP) zum Symmetriebruch während Neurulastadien führt. Dies wäre ein Tag später in der Entwicklung als vom ?Ionen-Fluss? Modell vorgeschlagen. In dieser Arbeit wurde die Funktion von ATP4a während des Symmetriebruchs nochmals näher untersucht. Die erhaltenen Ergebnisse legten eine symmetrische Verteilung und Funktion von ATP4 während der LR-Entwicklung nahe. Es konnte gezeigt werden, dass die Funktion von ATP4a in zwei Wnt-abhängigen Signalprozessen für die Entstehung des linksgerichteten Flüssigkeitsstroms benötigt wurde: (1.) Für die Wnt/b-Catenin-abhängige Expression von Foxj1 während der Gastrulation, und (2.) für die Wnt/PCP-abhängige (planare Zellpolarität) posteriore Positionierung von motilen Cilien während der Neurulation. Diese Daten stellten die ?Ionen-Fluss?-Hypothese in Frage und unterstützten die Idee eines konservierten Symmetriebruch-Mechanismus in Wirbeltieren, welcher ATP4- und Cilien-abhängig ist. Zudem wurden die Funktionen von weiteren Komponenten des Wnt-Signalwegs währen der Entstehung der GRP untersucht: Der Rezeptor Frizzled 8 (Fz8) und b-Catenin wurden für die Expression von Foxj1 während der Gastrulation benötigt. Funktionsverlust des non-kanonischen Liganden Xwnt11b hingegen störte die Morphogenese der GRP, die posteriore Ausrichtung von motilen Cilien, sowie die Expression von Coco und Xnr1 in somitischen Zellen der GRP. Dies legte nahe, dass neben Wnt/PCP die Aktivität weiterer non-kanonischer Signalzweige des Wnt-Signalweges für die LR-Entwicklung notwendig waren. ATP4-abhängige Wnt-Signalaktivität war auch für die Expression von Foxj1 und die Entstehung motiler Cilien in anderen ciliierten Epithelien während der Entwicklung von Xenopus notwendig: z.B. in der Haut, der neuralen Bodenplatte und im Ependym. In der Bodenplatte des Neuralrohrs wurde b-Catenin dem Hedgehog-Signalweg nachgeschaltet für die Foxj1 Expression benötigt. Im mucociliären Epithel der Haut wurden ATP4a und Wnt/b-Catenin gebraucht, nachdem die Zellen über den Notch/Delta-Signalweg spezifiziert wurden. Diese Art der Regulation wurde auch in einem neuen Zelltyp serotonerger Zellen beobachtet, welcher hier mittels morphologischer Analyse, Analyse der Genexpression und anhand der Reaktion auf Manipulation des Notch/Delta-Signalwegs charakterisiert wurde. Zusammenfassend kann gesagt werden, dass die in dieser Dissertation vorgelegten Daten für eine evolutionäre Konservierung der Funktionen von ATP4a und des Wnt-Signalweges beim Symmetriebruch der Wirbeltiere sprechen, sowie eine Verbindung zwischen ATP4a und Wnt bei der Foxj1-abhängigen Entstehung von Cilien in Xenopus herstellen

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

Has the war in Ukraine changed Europeans’ preferences on refugee policy? Evidence from a panel experiment in Germany, Hungary and Poland

The Russian invasion of Ukraine in early 2022 resulted in the largest refugee crisis in Europe since WWII. Using a unique panel conjoint experiment on refugee policy preferences carried out in Germany, Poland and Hungary just before and after the onset of the war in Ukraine, we show a heterogenous response to the influx of refugees from Ukraine across the three countries: no change of policy preferences in Germany, moderate change in Hungary and a significant change in Poland. Our results have direct implications for the development of a common EU asylum policy, as even though the countries persistently diverge on the preferred mode of asylum seekers’ allocation, with Germans favouring relocation and Poland and Hungary the status quo, the results highlight the scope for consensus rooted in shared preference for the asylum seekers’ unrestricted access to the labour market. This dimension consistently emerges as the most important policy dimension in all three countries before and after the outbreak of war

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

ΔN-Tp63 mediates Wnt/β-catenin-induced inhibition of differentiation in basal stem cells of mucociliary epithelia

Mucociliary epithelia provide a first line of defense against pathogens. Impaired regeneration and remodeling of mucociliary epithelia are associated with dysregulated Wnt/beta-catenin signaling in chronic airway diseases, but underlying mechanisms remain elusive, and studies yield seemingly contradicting results. Employing the Xenopus mucociliary epidermis, the mouse airway, and human airway Basal cells, we characterize the evolutionarily conserved roles of Wnt/beta-catenin signaling in vertebrates. In multiciliated cells, Wnt is required for cilia formation during differentiation. In Basal cells, Wnt prevents specification of epithelial cell types by activating Delta N-TP63, a master transcription factor, which is necessary and sufficient to mediate the Wnt-induced inhibition of specification and is required to retain Basal cells during development. Chronic Wnt activation leads to remodeling and Basal cell hyperplasia, which are reversible in vivo and in vitro, suggesting Wnt inhibition as a treatment option in chronic lung diseases. Our work provides important insights into mucociliary signaling, development, and disease

The Alternative Splicing Regulator Tra2b Is Required for Somitogenesis and Regulates Splicing of an Inhibitory Wnt11b Isoform

Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping), 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism