BackgroundCirculation 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.MethodsGene 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.ResultsRNA 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.ConclusionsMotile 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.

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.

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.

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.

BackgroundPark2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. The purpose of the present study was to analyze (1) the expression patterns of PACRG during vertebrate embryogenesis, and (2) whether the PACRG protein was required for left-right (LR) axis specification through cilia-driven leftward flow in Xenopus laevis.MethodsPACRG cDNAs were cloned and expression was analyzed during early embryonic development of Xenopus, mouse, rabbit and zebrafish. Antisense morpholino oligonucleotide (MO) mediated gene knockdown was applied in Xenopus to investigate LR development at the level of tissue morphology, leftward flow and asymmetric marker gene expression, using timelapse videography, scanning electron microscopy (SEM) and whole-mount in situ hybridization. Results were statistically evaluated using Wilcoxon paired and χ2 tests.ResultsPACRG mRNA expression was found in cells and tissues harboring cilia throughout the vertebrates. Highly localized expression was also detected in the brain. During early development, PACRG was specifically localized to epithelia where leftward flow arises, that is, the gastrocoel roof plate (GRP) in Xenopus, the posterior notochord (PNC) in mammals and Kupffer’s vesicle (KV) in zebrafish. Besides its association with ciliary axonemes, subcellular localization of PACRG protein was found around the nucleus and in a spotty pattern in the cytoplasm. A green fluorescent protein (GFP) fusion construct preferentially labeled cilia, rendering PACRG a versatile marker for live imaging. Loss-of-function in the frog resulted dose dependently in LR, neural tube closure and gastrulation defects, representing ciliary and non-ciliary functions of PACRG.ConclusionsThe PACRG protein is a novel essential factor of cilia in Xenopus.