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Characterization of Claudin-Dependent Morphogenetic Events During Neural Tube Closure and the Impact of CLDN Variants

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Characterization of Claudin-Dependent Morphogenetic Events During Neural Tube Closure and the Impact of CLDN Variants Characterization of Claudin-dependent morphogenetic events during neural tube closure and the impact of CLDN variants Amanda Baumholtz Department of Human Genetics McGill University, Montreal, Canada February 2018 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Amanda Baumholtz 2018 ABSTRACT The claudin family of tight junction proteins regulates paracellular permeability, apical- basal cell polarity, and cell adhesion and their cytoplasmic C-termini interact with the actin cytoskeleton. Through these activities, claudins have the potential to coordinate cell and tissue behaviors during epithelial morphogenesis. We previously showed that a subset of claudins is differentially expressed between the neural and non-neural epithelium during neural tube closure. This led to my hypothesis that these domains of claudin expression correlate to claudin function during neural tube morphogenesis and, if true, I predicted that deleterious missense mutations in CLDN genes would contribute to increased susceptibility to human neural tube defects (NTDs). I showed that selective removal of two of the eleven claudins expressed in the neural ectoderm of chick embryos, Cldn4 and -8, caused open NTDs due to defective convergent extension and failure of apical constriction at the neural plate midline. The failure of these morphogenetic events appears to be due to aberrant protein localization to the apical surface. In contrast, removing only Cldn3 from the non-neural ectoderm affected the epithelial remodeling events required for fusion of the dorsal tips of the neural folds to form the closed neural tube and continuous overlying layer of non-neural ectoderm. Claudin-depleted mouse embryos also exhibited NTDs. Sequence analysis of 125 patients with open spinal NTDs identified nine rare and five novel missense variants in nine CLDN genes. Functional validation studies revealed that overexpression of CLDN19 I22T and E209G, but not wild-type CLDN19, caused open NTDs in chick embryos due to defects in neural fold fusion and convergent extension, respectively. My data indicate that claudins play an evolutionary conserved role in vertebrate neural tube closure and that deleterious missense mutations in CLDN genes may contribute to human NTDs by impeding critical phases of neural tube closure. Furthermore, my data indicate that the combination of claudins expressed in an epithelial cell layer creates distinct compartments that regulate intracellular signaling events at the apical surface of epithelial cells to influence epithelial morphogenesis. i RÉSUMÉ Les claudines sont des composantes intégrales des jonctions serrées et qui régulent la perméabilité paracellulaire, la polarité cellulaire apicale-basale, et l’adhésion cellulaire. De plus, le domaine C-terminale des claudines se lie au cytosquelette d’actine. Par l’entremise de ces activités, les claudines ont le potentiel de coordonner les comportements des cellules et des tissus pendant la morphogenèse épithéliale. Précédemment, nous avons démontré que l’expression des membres de la famille des claudines est différente dans le neuroépithélium comparé à l’épithélium non-neural pendant la fermeture du tube neural. Cette observation nous a mené à formuler l`hypothèse suivante: les patrons d’expression des claudines dans l’épithélium correspondent aux fonctions des claudines pendant la formation du tube neural. Si tel est le cas, des mutations pathogéniques dans les CLDN contribueraient aux facteurs génétiques augmentent le risque d’anomalies de fermeture du tube neural. Nous avons démontré que la suppression sélective de deux des onze claudines exprimées dans les cellules neuroectodermiques de l’embryon de poulet cause des anomalies de fermeture du tube neural dues aux défauts de l’extension convergente et à la constriction apicale des cellules au centre de la plaque neurale. L’échec de mouvements morphogéniques semble être causé par la relocalisation des protéines à la surface apicale. La suppression de Cldn3 dans les cellules non-neuroectodermiques cause des anomalies de fermeture du tube neural dues au fait que les plis neuraux, ainsi que l`ectoderme non-neurale, ne fusionnent pas. La suppression de certaines claudines dans les embryons de souris cause un défaut de fermeture du tube neural. Le séquençage de 125 patients avec des anomalies de fermeture du tube neural à l’extrémité caudale a identifié neuf changements nucléiques rares et cinq changements non-rapportés dans neuf CLDN. Les études fonctionnelles ont montré que la surexpression du changement protéique I22T et E209G dans CLDN19, mais pas dans CLDN19 sauvage, ne permettait pas la fermeture du tube neural dans les embryons de poulet. Ceci est dû au défaut de fusionnement des plis neuraux et à l’extension convergente, respectivement. Ces résultats suggèrent que les claudines jouent un rôle essentiel dans la fermeture du tube neural chez les vertébrés et que les changements faux-sense pathogéniques dans ces gènes peuvent contribuer aux anomalies de fermeture du tube neural chez les humains en bloquant certaines phases critiques lors de la fermeture du tube neural. ii De plus, la combinaison de claudines exprimée par les tissus épithéliaux crée des microenvironnements qui servent à coordonner les évènements intracellulaires au niveau du pôle apical. iii TABLE OF CONTENTS ABSTRACT .............................................................................................................................. i RÉSUMÉ ................................................................................................................................. ii LIST OF FIGURES ............................................................................................................. xiii CHAPTER I: INTRODUCTION AND LITERATURE REVIEW ................................... 1 1.1 OVERVIEW AND RATIONALE FOR STUDY ........................................................ 2 1.2 TIGHT JUNCTIONS ..................................................................................................... 3 1.2.1 Identification of tight junctions ................................................................................. 3 1.2.2 Evolutionary conservation of tight junctions ............................................................ 4 1.2.3 Tight junction transmembrane proteins .................................................................... 4 1.2.3.1 Claudins ................................................................................................................ 5 1.2.3.2 Occludin ................................................................................................................ 5 1.2.3.3 Tricellulin ............................................................................................................. 6 1.2.3.4 MarvelD3 .............................................................................................................. 7 1.2.3.5 Junctional adhesion molecules ............................................................................. 7 1.2.4 Tight junction cytoplasmic proteins .......................................................................... 8 1.2.4.1 ZO proteins ........................................................................................................... 8 1.2.4.2 Cingulin/paracingulin ......................................................................................... 10 1.2.4.3 MUPP1 ............................................................................................................... 10 1.2.5 Formation of tight junctions.................................................................................... 10 1.2.5.1 Crumbs polarity complex ................................................................................... 11 1.2.5.2 Par polarity complex ........................................................................................... 11 1.2.6 Regulation of tight junction assembly/disassembly ................................................ 12 1.2.7 Functions of tight junctions .................................................................................... 12 1.2.7.1 The gate functions of tight junctions .................................................................. 13 1.2.7.2 The fence function of tight junctions .................................................................. 14 1.2.7.3 Tight junctions and adhesion .............................................................................. 14 1.2.7.4 Tight junctions and cell proliferation and gene expression ................................ 15 1.3 THE CLAUDIN FAMILY OF TIGHT JUNCTION PROTEINS........................... 16 1.3.1 Identification of claudins ........................................................................................ 16 iv 1.3.2 Structure of claudins ............................................................................................... 16 1.3.3 Claudin-claudin interactions ................................................................................... 17 1.3.4 Post-translational modifications .............................................................................. 18 1.3.4.1 Phosphorylation .................................................................................................. 18 1.3.4.2 Palmitoylation ..................................................................................................... 19 1.3.5 Genomic organization and evolutionary conservation of claudins ........................
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