BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) is in the family of structural renal tract birth defects. CAKUT is the major cause of chronic kidney disease and renal failure in children and adults <40 years of age. ROBO2 is a receptor for the SLIT2 ligand. ROBO2/SLIT2 signaling has been shown to play important roles in neuronal migration and in early renal tract development. Our laboratory has recently identified ROBO2 as a novel CAKUT-causing gene. So far, total 26 ROBO2 mutations have been identified in patients with CAKUT. However, most of these mutations are missense amino acid substitutions and their functional significances are unclear, although they are predicted to be disease-causing by several bioinformatics prediction software. OBJECTIVE: To clarify uncertainties and confusions in the CAKUT field regarding the causality for ROBO2 missense mutations, we performed functional analysis of a ROBO2 missense mutation p.G114W (c.340G>T) that was identified in a CAKUT family. This p.G114W ROBO2 mutation is located in the first Ig domain of the ROBO2 extracellular region. The ROBO2 first Ig domain is the binding site for ligand SLIT2 that is required for ROBO2-SLIT2 signaling chemorepulsive activity in neuronal migration. We hypothesize that this p.G114W ROBO2 mutation would disrupt the SLIT2-ROBO2 binding and compromise its chemorepulsive activity in a sensitive functional neuronal migration assay. METHODS: Site directed mutagenesis was used to introduce c.340G>T point mutation into a ROBO2 cDNA fusion construct that contains the first Ig domain. Point mutation was verified using Sanger sequencing. Mutant ROBO2 cDNA and wildtype control constructs were purified using Qiagen Midiprep kit and transfected in HEK cells via calcium phosphate co-precipitation. The conditioned medium (CM) containing ROBO2 fusion proteins were analyzed by Western Blot. Neuronal migration assays were performed using postnatal anterior subventricular zone (SVZa) tissue explants that were isolated from postnatal day 1 to 5 (P1-5) Sprague-Dawley rat brain. RESULTS: By Sanger sequencing, we verified the c.340G>T point mutation in the ROBO2 cDNA fusion construct. By GFP fluorescence and Western blot analysis, we found abundant expressions of ROBO2 fusion protein in the conditioned medium of transfected HEK cells. In SVZa neuronal migration assays, we found that, when compared to the wild type fusion protein, the mutant ROBO2 fusion protein with the p.G114W amino acid substitution lost its function to block SLIT2-medicated inhibition of neuronal migration at both 50% conditioned medium and 100% conditioned medium concentrations. CONCLUSION: Our results show that ROBO2 p.G114W is a loss-of-function mutation disrupting normal SLIT2-ROBO2 chemorepulsive activity on SVZa neuronal migration, suggesting that the presence of this missense mutation compromises SLIT2-ROBO2 signaling and contributes to the development of CAKUT. / 2020-07-03T00:00:00Z

Abnormal kidney development is a relatively prevalent health issue; however, the genetic basis is mostly unknown. The aim of this thesis is to identify genes important in kidney development and disease and to study their molecular functions. We hypothesized that human diseases associated with kidney anomalies can uncover novel genes important in kidney development and disease. The thesis is divided into three independent projects that examined three genes (i.e. Zeb2, Ilk, Robo2) at three stages of mouse kidney development: nephrogenesis, glomerular podocyte, and early ureteric bud outgrowth. In the first project, we identified Zeb2, a gene encoding the zinc finger E-box binding homeobox 2 transcription factor that is mutated in the Mowat Wilson syndrome, as a novel gene important in nephrogenesis. Zeb2 conditional knockout mice (Zeb2 cKO) develop glomerulocystic kidney disease with many atubular glomeruli and decreased expression of proximal tubular markers before cyst formation. These data suggest that abnormal nephrogenesis leads to the congenital atubular glomeruli and primary glomerular cysts in the Zeb2 cKO mice. This study implies that ZEB2 is a novel candidate gene for glomerular cystic disease in patients. Additionally we found that Pkd1, the gene mutated in autosomal dominant polycystic kidney disease, is upregulated in non-cystic glomeruli and knockout of one copy of the Pkd1 gene exacerbates the cystic phenotype of the Zeb2 cKO mice. These findings suggest a genetic interaction between Zeb2 and Pkd1 and that Zeb2 might be a novel PKD1 modifier. In the second project, we studied the roles of integrin-linked kinase (ILK) and roundabout 2 (ROBO2) in glomerular podocytes. We found that ILK and ROBO2 form a protein complex, and that loss of Robo2 improves survival and alleviates the podocyte and basement membrane abnormalities seen in Ilk knockout mice. In the third project, using microarray gene expression analysis, we found lower gene expression levels of extracellular matrix proteins during early ureteric bud outgrowth in the Robo2 homozygous knockout embryos as compared to wild type controls. These findings suggest that ROBO2 may regulate extracellular matrix components in the kidney. In conclusion, we found a new role for Zeb2 in nephrogenesis, and identified a novel function of Robo2 in regulating extracellular matrix gene expression in podocytes and during early kidney development. / 2017-11-03T00:00:00Z

Les commissures forment un ensemble de connexions nerveuses assurant la communication entre les neurones de chaque hémi partie du système nerveux central des bilatériens. Au cours du développement embryonnaire, les axones des neurones commissuraux sont guidés au travers de la ligne médiane délimitant ces deux parties. Plusieurs sources de signaux de guidage attractifs et répulsifs agissent de concert pour organiser les trajectoires de ces axones. Dans la moelle épinière, les axones commissuraux traversent la ligne médiane dans un territoire ventral, la plaque du plancher (PP). Au cours de la traversée de la PP, ils acquièrent une sensibilité à des signaux répulsifs exprimés par ce territoire qui leur empêchent de rebrousser le chemin et qui les poussent hors de la PP. Plusieurs couples ligands/récepteurs médient ces forces répulsives mais les mécanismes qui sous-tendent l'acquisition de la sensibilité aux signaux répulsifs restent encore peu connus. Par exemple on ignore si les axons se sensibilisent à tous les signaux répulsifs en même temps, quand précisément ce switch de réponse se fait, et les contributions précises de chacun de ces signaux. Une spécificité fonctionnelle est suggérée par l'analyse des phénotypes d'invalidation des gènes codant pour ces récepteurs chez la souris ou encore par des manipulations d'expression chez l'embryon de poulet. L'objectif de mes travaux de thèse a été de tester l'hypothèse selon laquelle la génération de spécificités fonctionnelles pourrait résulter de contrôles précis et distincts de la dynamique spatiale et temporelle des récepteurs de guidage à la surface du cône de croissance. J'ai tout d'abord développé un dispositif de vidéomicroscopie adapté à l'enregistrement de cônes de croissance accomplissant la traversée de la PP, sur des moelles épinières en configuration de «livre ouvert». Afin de visualiser l'adressage à la surface du cône de croissance, j'ai exploité une forme de GFP sensible au pH, dont les propriétés de fluorescence à pH neutre permettent un suivi spécifique du pool de surface des protéines (Nawabi et al., 2010; Delloye-Bourgeois et al, 2014). J'ai utilisé ce paradigme pour comparer la dynamique temporelle de 4 récepteurs médiant les réponses aux divers signaux répulsifs de la PP: Nrp2, Robo1, Robo2 et PlxnA1. Les vecteurs d'expression de ces formes pHLuo de récepteurs ont été introduits dans les neurones commissuraux de la moelle épinière d'embryon de poulet par électroporation in ovo. Par des approches de microscopie à super-résolution sur les livres-ouverts, j'ai aussi étudié la distribution spatiale des récepteurs répulsifs à la surface des cônes de croissances au cours de la traversée. L'ensemble de ces expériences a pu démontrer que les récepteurs sont adressés à la membrane à différents temps de la navigation de la PP et occupent, de plus, des domaines distincts du cône de croissance. J'ai ensuite adapté la technique d'électroporation à la moelle épinière d'embryon de souris. Ces expériences ont montré que les séquences temporelles observées chez le poulet sont conservées chez la souris. J'ai également réintroduit le récepteur Robo1 dans une lignée de souris présentant une invalidation des récepteurs Robo1/2 et montré que l'altération de la traversée de la PP caractéristique de cette lignée est abolie dans la population d'axones capables d'adresser le récepteur Robo1 à la membrane. Au final, mes résultats démontrent que les axones commissuraux ne sont pas sensibilisés aux signaux répulsifs par la mise en œuvre d'un programme général. Au contraire, les récepteurs de guidage possèdent des profils de dynamiques temporelles spécifiques, et des domaines de distribution distincts dans le cône de croissance. Le contrôle de la dynamique d'adressage représente ainsi un mécanisme permettant de discriminer des signaux concomitants, en les fonctionnalisant à différents temps de la navigation de la moelle épinière / During embryonic development, commissural axons are guided through the midline, crossing from one side of the CNS to the other one at specific time points and positions to project onto contralateral neurons. Several sources of attractive cues regulate their navigation. In addition, repulsive forces act at different steps to keep the axons along their path. In the developing spinal cord, commissural axons cross the midline in a ventral territory, the floor plate (FP). Commissural axons gain sensitivity to repellents present in the FP after their crossing. The setting of these novel properties is necessary for preventing the axons to cross back and also for pushing them towards FP exit. Various ligand/receptor couples have been reported to mediate these repulsive forces. Whether commissural axons gain response to all the repulsive cues at the same time is not known. Whether these repulsive cascades have specific functions is suggested by different outcome of their invalidation in mouse models, but how are set these differences also remains unknown. We hypothesized that the generation of functional specificities could be achieved though specific controls of the spatial and temporal dynamics of guidance receptors at the growth cone surface. During my PhD, I developed a set up for time-lapse imaging of “open book” spinal cords, to monitor the dynamics of guidance receptors in axons experiencing native guidance decisions across the midline. To visualize their cell surface sorting, receptors were fused to the pH-sensitive GFP, pHLuorin, whose fluorescence at neutral pH reports membrane protein pools (Nawabi et al, 2010; Delloye-Bourgeois et al, 2014), and were expressed in spinal commissural neurons through in ovo electroporation. This paradigm revealed striking differences in the temporal dynamics of Nrp2, Robo1, Robo2 and PlexinA1, the receptors known to mediate the responsiveness to the major midline repellents referenced in vertebrates: Slit-Ns, Slit-Cs and Semaphorin3B. Moreover, using super-resolution microscopy, I could evidence that PlexinA1 and Robo1 are sorted in distinct subdomains of commissural growth cones navigating the floor plate. I also introduced the pHLuo-tagged receptors in the mouse embryo. These experiments showed that the temporal sequences established in the chick are conserved in the mouse, and that FP crossing in Robo1/2 mutant embryos was rescued in growth cones that could achieve cell surface sorting of Robo1. Thus, my results show that guidance receptors for midline repellents have highly specific spatial and temporal dynamics. The generation of a temporal sequences of cell surface sorting thus represents a mechanism whereby commissural growth cones discriminate concomitant signals by functionalizing them at different timing of their spinal cord navigation

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22599号 / 生博第432号 / 新制||生||57(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 鈴木 淳, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Glomerular derangement is the major feature of a diverse array of kidney disorders that lead to end-stage kidney disease (ESKD). Podocyte dysfunction is central to the underlying pathophysiology of many common glomerular diseases, including diabetic nephropathy (DN), focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), and genetic forms of nephrotic syndrome, and is associated with heavy proteinuria. Loss of podocyte foot process structure, or effacement, is the key feature of podocyte injury in these proteinuric glomerular disorders, also called "podocytopathies". However, the degree of effacement can vary: For instance, it is very disseminated (diffuse) in minimal change disease, but more variable (segmental) in focal segmental glomerulosclerosis. Recent work has shown that nephrin, ROBO2, and SLIT2 are proteins implicated in these podocyte foot process effacement and podocytopathy. We sought to evaluate changes in the expression of these proteins using immunofluorescence microscopy, and the degree of foot process effacement in podocytopathies using ultrastructural morphometry. In podocytopathies, we saw increased expression of ROBO2 and decreased expression of nephrin indicating that, upregulation of ROBO2 may lead to podocyte injury and podocyte injury may result in loss of nephrin. In addition, SLIT2, which binds ROBO2, was found in tubules and in glomeruli. A higher degree of geometric mean foot process width was vii observed in podocytopathies as compared to normal kidney. These findings can be used in the clinical setting to diagnose and monitor disease treatment. / 2024-02-07T00:00:00Z