Mutational and functional analyses of Bardet-Biedl syndrome

This thesis investigates some of the underlying causes of Bardet-Biedl syndrome, a leading example of emerging ciliopathies. Bardet-Biedl syndrome (BBS) is a heterogeneous disorder with primary features that include age-related retinal dystrophy, obesity, Polydactyly, renal dysplasia, reproductive tract anomalies and cognitive impairment. To date 12 loci have been found to be causative for the disease (BBS1-12) and evidence suggests that aspects of the BBS phenotype may result from defective ciliogenesis/basal body function. The aims of this project were to identify new genes involved with the disease, analyse the phenotype of the Bbs6"7" mouse and further elucidate the mechanism of the disease in a zebrafish model. Pathogenic sequence mutations identified in candidate genes aided the identifictaion of BBS3 and BBS5. Further candidate genes were identified through the mapping of translocation breakpoints in a BBS patient. A yeast two-hybrid screen, using Bbs6 as bait, revealed several potential protein interactions. Phenotypical analysis of the Bbs&A mouse showed features comparable to BBS patients and other Bbs null mice, confirming its value as a model for further study. Further investigations of BBS proteins in the cochlea suggested a role beyond that of cilia and basal body function, namely that BBS proteins play distinct roles in the processes of microtubule nucleation and growth. Observed disruption of cochlear stereociliary bundles, along with other phenotypes associated with planar cell polarity (PCP) mutants, implied cilia might be involved in PCP signalling. This observation was supported by further analysis in the zebrafish. Disruption of bbs8 resulted in similar phenotypes to other PCP mutants, and laterality defects, thought to arise from defective cilia function at the Kupffer's Vesicle, were enhanced on a PCP mutant background. The results presented in this thesis pave the way for further investigation with a view to broadening the knowledge of the developmental processes behind BBS and also the processes behind the development of other signaling pathways, tissues and organ systems

Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo

Mutational and Functional Analyses of Bardet-Biedi Syndrome

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Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a

Planar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six “core PCP” components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. We recently demonstrated a role of Prickle1, a core PCP molecule in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, we generated three mutant alleles in mice. We show that the complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Curiously, Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a or Ror2 gene defects, prompting us to explore an association of Prickle1 with the Wnt pathway. We show that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. Our studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome

Generation of an inducible RPE-specific Cre transgenic-mouse line.

The retinal pigment epithelium (RPE) is an epithelial monolayer in the back of the vertebrate eye. RPE dysfunction is associated with retinal degeneration and blindness. In order to fully understand how dysregulation affects visual function, RPE-specific gene knockouts are indispensable. Since the currently available RPE-specific Cre recombinases show lack of specificity or poor recombination, we sought to generate an alternative. We generated a tamoxifen-inducible RPE-specific Cre transgenic mouse line under transcriptional control of an RPE-specific Tyrosinase enhancer. We characterized the Cre-mediated recombinant expression by crossing our RPE-Tyrosinase-CreErT2 mouse line with the tdTomato reporter line, Ai14. Detected fluorescence was quantified via high-content image analysis. Recombination was predominantly observed in the RPE and adjacent ciliary body. RPE flatmount preparations revealed a high level of recombination in adult mice (47.25-69.48%). Regional analysis of dorsal, ventral, nasal and temporal areas did not show significant changes in recombination. However, recombination was higher in the central RPE compared to the periphery. Higher levels of Cre-mediated recombinant expression was observed in embryonic RPE (~83%). Compared to other RPE-specific Cre transgenic mouse lines, this newly generated RPE-Tyrosinase-CreErT2 line shows a more uniform and higher level of recombination with the advantage to initiate recombination in both, prenatal and postnatal animals. This line can serve as a valuable tool for researches exploring the role of individual gene functions, in both developing and differentiated RPE

Behavioral Phenotyping of Bbs6 and Bbs8 Knockout Mice Reveals Major Alterations in Communication and Anxiety

The primary cilium is an organelle with a central role in cellular signal perception. Mutations in genes that encode cilia-associated proteins result in a collection of human syndromes collectively termed ciliopathies. Of these, the Bardet-Biedl syndrome (BBS) is considered one of the archetypical ciliopathies, as patients exhibit virtually all respective clinical phenotypes, such as pathological changes of the retina or the kidney. However, the behavioral phenotype associated with ciliary dysfunction has received little attention thus far. Here, we extensively characterized the behavior of two rodent models of BBS, Bbs6/Mkks, and Bbs8/Ttc8 knockout mice concerning social behavior, anxiety, and cognitive abilities. While learning tasks remained unaffected due to the genotype, we observed diminished social behavior and altered communication. Additionally, Bbs knockout mice displayed reduced anxiety. This was not due to altered adrenal gland function or corticosterone serum levels. However, hypothalamic expression of Lsamp, the limbic system associated protein, and Adam10, a protease acting on Lsamp, were reduced. This was accompanied by changes in characteristics of adult hypothalamic neurosphere cultures. In conclusion, we provide evidence that behavioral changes in Bbs knockout mice are mainly found in social and anxiety traits and might be based on an altered architecture of the hypothalamus

Primary Cilia Structure Is Prolonged in Enteric Neurons of 5xFAD Alzheimer’s Disease Model Mice

Neurodegenerative diseases such as Alzheimer’s disease (AD) have long been acknowledged as mere disorders of the central nervous system (CNS). However, in recent years the gut with its autonomous nervous system and the multitude of microbial commensals has come into focus. Changes in gut properties have been described in patients and animal disease models such as altered enzyme secretion or architecture of the enteric nervous system. The underlying cellular mechanisms have so far only been poorly investigated. An important organelle for integrating potentially toxic signals such as the AD characteristic A-beta peptide is the primary cilium. This microtubule-based signaling organelle regulates numerous cellular processes. Even though the role of primary cilia in a variety of developmental and disease processes has recently been recognized, the contribution of defective ciliary signaling to neurodegenerative diseases such as AD, however, has not been investigated in detail so far. The AD mouse model 5xFAD was used to analyze possible changes in gut functionality by organ bath measurement of peristalsis movement. Subsequently, we cultured primary enteric neurons from mutant mice and wild type littermate controls and assessed for cellular pathomechanisms. Neurite mass was quantified within transwell culturing experiments. Using a combination of different markers for the primary cilium, cilia number and length were determined using fluorescence microscopy. 5xFAD mice showed altered gut anatomy, motility, and neurite mass of enteric neurons. Moreover, primary cilia could be demonstrated on the surface of enteric neurons and exhibited an elongated phenotype in 5xFAD mice. In parallel, we observed reduced β-Catenin expression, a key signaling molecule that regulates Wnt signaling, which is regulated in part via ciliary associated mechanisms. Both results could be recapitulated via in vitro treatments of enteric neurons from wild type mice with A-beta. So far, only a few reports on the probable role of primary cilia in AD can be found. Here, we reveal for the first time an architectural altered phenotype of primary cilia in the enteric nervous system of AD model mice, elicited potentially by neurotoxic A-beta. Potential changes on the sub-organelle level—also in CNS-derived neurons—require further investigations

The Interplay between Nutrition, Innate Immunity, and the Commensal Microbiota in Adaptive Intestinal Morphogenesis

The gastrointestinal tract is a functionally and anatomically segmented organ that is colonized by microbial communities from birth. While the genetics of mouse gut development is increasingly understood, how nutritional factors and the commensal gut microbiota act in concert to shape tissue organization and morphology of this rapidly renewing organ remains enigmatic. Here, we provide an overview of embryonic mouse gut development, with a focus on the intestinal vasculature and the enteric nervous system. We review how nutrition and the gut microbiota affect the adaptation of cellular and morphologic properties of the intestine, and how these processes are interconnected with innate immunity. Furthermore, we discuss how nutritional and microbial factors impact the renewal and differentiation of the epithelial lineage, influence the adaptation of capillary networks organized in villus structures, and shape the enteric nervous system and the intestinal smooth muscle layers. Intriguingly, the anatomy of the gut shows remarkable flexibility to nutritional and microbial challenges in the adult organism