Analsysis of the ENU-generated mouse mutant cbs: Primary cilia play a crucial role in cortical development

In the following study the mouse mutant cobblestone (cbs) concerning the development of the forebrain was analyzed. The cbs mutation was uncovered by an ethyl-nitroso-urea (ENU) genetic screen, using a mouseline called tauGFP. At the beginning of the analysis of the cbs mutation, the phenotype of the cbs/cbs mouse mutant was already known, but because of the mutagenic ability of ENU, which causes random mutations, the affected gene was unclear. By applying the method of positional cloning the gene intraflagellar transport 88 (Ift88) was identified as the candidate gene. A detailed analysis of mRNA levels of Ift88 in the cbs/cbs mutant was undertaken by Northen Blot analysis as well as quantitative real-time RT-PCR. At the same time the Ift88 protein levels were also investigated by Western blot analysis. A complementation analysis by crossing cbs heterozygotes to mice heterozygous for a targeted deletion of the Ift88 gene (Ift88tm1.1Bky) (Haycraft et al., 2007) was done to ascertain, if the genetic defect in the cbs/cbs mutant is located in the Ift88 gene. In the represented study it could be shown that cbs is a hypomorphic allele of the gene Ift88, in which both Ift88 mRNA and protein levels are reduced by 70% to 80%, respectively. A detailed analysis by in situ hybridization (ISH) was followed, using different markers, which are specific for various areas of the developing telencephalon such as Ttr1 for the choroid plexus, Wnt2b for the cortical hem, as well as EphB1 and Lhx2 for the hippocampal anlage. Furthermore both the dorsal-ventral and rostral-caudal compartmental boundaries of the forebrain were investigated by ISH. cbs/cbs mutants display defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus. Furthermore mutants exhibit a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries of the forebrain, resulting in the intermixture of otherwise separated cell populations. It is further demonstrated that the proteolytic processing of Gli3 is reduced in the cbs/cbs mutant, leading to an accumulation of the full-length activator isoform. In addition the cbs/cbs mutant exhibits an upregulation of canonical Wnt signalling in the neocortex and in the caudal forebrain. Primary cilia, microtubule-based organelles that protrude from the surface of most cells of the vertebrate body, are dependent on Ift88 for their formation and maintenance. The ultrastructure and morphology of cilia of the ventricle of the cbs/cbs mutants was therefore simultaneously investigated by transmission and scanning electron microscopy. Surprisingly, examined cilia are still existing and intact in the cbs/cbs mutant. Taken together, these results indicate a fundamental role for primary cilia in the development of the forebrain

Positional Cloning of cobblestone, a Mouse Mutant Showing Major Defects in Brain Development, Identifies Ift88 as a Candidate Gene

The ENU-induced cobblestone (cbs) mouse mutant exhibits severe defects in fore- and midbrain development. Via genomic mapping, the causative mutation for the cbs-phenotype has previously been located on chromosome 14 in the proximity of the gene Ift88 (Intraflagellar transport, 88 kDa). The Ift88 protein is involved in intraflagellar transport and is required for genesis and maintenance of primary and motile cilia. In order to refine the genetic interval further, we performed fine-mapping analysis of the cbs mutant. The candidate region of the cbs mutation is shown to be situated in a region of 4.14 Mb on chromosome 14, containing the Ift88 gene, and excluding dozens of genes present in the roughly-mapped interval. However, neither sequencing of the core Ift88 promoter region nor of two conserved intronic sequences within Ift88 revealed any mutations, indicating that the responsible mutation lies in a transcriptional regulatory sequence within or near the Ift88 gene. Finally, other potential candidate genes in this genetic interval have been identified using synteny analysis on six other vertebrate genomes. This analysis is thus compatible with the idea that the mutation in cbs is located on the Ift88 gene, but it also allows the possibility of other candidate genes that lie near Ift88 to be involved in the phenotype

A Crucial Role for Primary Cilia in Cortical Morphogenesis

Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

The dynamic cilium in human diseases

Cilia are specialized organelles protruding from the cell surface of almost all mammalian cells. They consist of a basal body, composed of two centrioles, and a protruding body, named the axoneme. Although the basic structure of all cilia is the same, numerous differences emerge in different cell types, suggesting diverse functions. In recent years many studies have elucidated the function of 9+0 primary cilia. The primary cilium acts as an antenna for the cell, and several important pathways such as Hedgehog, Wnt and planar cell polarity (PCP) are transduced through it. Many studies on animal models have revealed that during embryogenesis the primary cilium has an essential role in defining the correct patterning of the body. Cilia are composed of hundreds of proteins and the impairment or dysfunction of one protein alone can cause complete loss of cilia or the formation of abnormal cilia. Mutations in ciliary proteins cause ciliopathies which can affect many organs at different levels of severity and are characterized by a wide spectrum of phenotypes. Ciliary proteins can be mutated in more than one ciliopathy, suggesting an interaction between proteins. To date, little is known about the role of primary cilia in adult life and it is tempting to speculate about their role in the maintenance of adult organs. The state of the art in primary cilia studies reveals a very intricate role. Analysis of cilia-related pathways and of the different clinical phenotypes of ciliopathies helps to shed light on the function of these sophisticated organelles. The aim of this review is to evaluate the recent advances in cilia function and the molecular mechanisms at the basis of their activity

The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating patterning of prethalamus and ventral telencephalon

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3-/- corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3-/- VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development

Crisis Communication in Sports

Durch inkorrekte, zu langsame oder überstürzte Krisenkommunikation kommt es jährlich zu schwerwiegenden ökonomischen Schäden und einem Reputationsverlust für Unternehmen und Sportorganisationen. Ein professionelles Krisenmanagement wird für Sportler und Sportorganisationen in der heutigen Zeit daher immer wichtiger. Die vorliegende Masterarbeit beschäftigt sich mit diesem Aufgabenfeld und analysiert den Krisenfall der Vergabe der Fußballweltmeisterschaft 2006 an Deutschland. Hierzu wird das Kommunikationsverhalten der beteiligten Personen des Organisationskomitees der Weltmeisterschaft und des Deutschen Fußballbundes aufgezeigt und bewertet. Zunächst werden die notwendigen theoretischen Grundlagen von Krisenmanagement, der Rolle der Medien im Krisenfall sowie die speziellen Rahmenbedingungen im Sport erläutert. Ziel der Arbeit ist es, Erfolgsfaktoren sowie Handlungsempfehlungen für ein professionelles Krisenmanagement abzuleiten und zugleich auf den Sport zu adaptieren, da aufgrund der hohen medialen Aufmerksamkeit und der daraus folgenden erhöhten Wahrnehmung der Öffentlichkeit die Kommunikation im Sport branchenbedingt häufig Krisenkommunikation ist

Poster by Sadie Willaredt

Designs by students in English 3005, Dr. Julie Campbellhttps://thekeep.eiu.edu/lib_comiccon_posters/1001/thumbnail.jp