La protéine de liaison aux ARN MEX-3 est un régulateur essentiel du développement embryonnaire chez le nématode Caenorhabditis elegans. Une famille de quatre gènes homologues à hMex-3 (dénommés hMex-3A, 3B, 3C et 3D) a été identifiée chez les mammifères par notre équipe. Afin de mieux comprendre la fonction physiologique in vivo des protéines Mex-3, nous avons invalidé le gène Mex-3B chez la souris. Cette approche expérimentale a révélé que Mex-3B est un acteur majeur de la spermatogenèse. Les souris mâles nullizygotes présentent une obstruction des tubes séminifères conduisant à une réduction importante du nombre des spermatozoïdes produits. L’ablation de Mex-3B ciblée à la cellule de Sertoli, cellule somatique essentielle à la fonction de l’épithélium séminifère, a permis d’établir que le phénotype testiculaire a pour origine une perturbation des propriétés biologiques de cette cellule. En effet, les cellules de Sertoli déficientes pour Mex-3B présentent des défauts de la phagocytose qui conduisent à une élimination défectueuse des corps résiduels au cours de la spemiogenèse. L’exploration des mécanismes moléculaires impliqués a montré que Mex-3B contrôle la phagocytose via la régulation de l’activité et de la localisation membranaire de Rap1GAP, une protéine qui régule négativement la petite protéine G Rap1. En accord avec ces données, l’absence de Mex-3B provoque une déstabilisation de la barrière hémato-testiculaire due à un défaut de localisation à la membrane plasmique des molécules de jonction connexine 43 et N-Cadhérine, protéines dont la translocation et la stabilité dépendent de Rap1. En conclusion, mes travaux de thèse ont permis de mettre en évidence un rôle clé de Mex-3B dans le contrôle spatial de la voie de signalisation Rap1 au cours de la spermatogenèse / The RNA binding-protein MEX-3 is a post-transcriptional regulator involved in early embryogenesis of the nematode Caenorhabditis elegans. We have recently reported the characterization of a novel family of four mammalian genes homologous to hMex-3 (called hMex-3A, 3B, 3C and 3D). To gain insight into the biological functions of these proteins in vivo, we disrupted the Mex-3B gene in mice. Using this experimental approach, we found that Mex-3B is as a major regulator of spermatogenesis. We observed that male Mex-3B null mice hypofertile and present an obstruction of seminiferous epithelium. Phagocytic properties of Sertoli cells were impaired, thus impeding the clearance of residual bodies released during spermiogenesis. Exploration of the underlying molecular mechanisms revealed that Mex-3B regulates phagocytosis through the activation and the transport at the peripheral membrane of Rap1GAP, a protein that downregulates the small G protein Rap1. Consistently, the Rap1-dependent recruitment of the junction proteins, connexin 43 and N-Cadherin at the cell surface was compromised in Mex-3B deficient mice. In conclusion, my work highlights a key role gor Mex-3B in the spatial control of Rap1 signaling during spermatogenesis

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During development, cells consume energy, divide, rearrange, and die. Bulk properties such as viscosity and elasticity emerge from cell-scale mechanics and dynamics. Order appears, for example in patterns of hair outgrowth, or in the predominately hexagonal pattern of cell boundaries in the wing of a fruit fly. In the past fifty years, much progress has been made in understanding tissues as living materials. However, the physical mechanisms underlying tissue-scale behaviour are not completely understood. Here we apply theories from statistical physics and fluid dynamics to understand mechanics and order in two-dimensional tissues. We restrict our attention to the mechanics and dynamics of cell boundaries and vertices, and to planar polarity, a type of long-ranged order visible in anisotropic patterns of proteins and hair outgrowth. Our principle tool for understanding mechanics and dynamics is a vertex model where cell shapes are represented using polygons. We analytically derive the ground-state diagram of this vertex model, finding it to be dominated by the geometric requirement that cells be polygons, and the topological requirement that those polygons tile the plane. We present a simplified algorithm for cell division and growth, and furthermore derive a dynamic equation for the vertex model, which we use to demonstrate the emergence of quasistatic behaviour in the limit of slow growth. All our results relating to the vertex model are consistent with and build off past calculations and experiments. To investigate the emergence of planar polarity, we develop quantification methods for cell flow and planar polarity based on confocal microscope images of developing fly wings. We analyze cell flow using a velocity gradient tensor, which is uniquely decomposed into terms corresponding to local compression, shear, and rotations. We argue that a pattern in an inhomogeneously flowing tissue will necessarily be reorganized, motivating a hydrodynamic theory of polarity reorientation. Using such a coarse-grained theory of polarity reorientation, we show that the quantified patterns of shear and rotation in the wing are consistent with the observed polarity reorganization, and conclude that cell flow reorients planar polarity in the wing of the fruit fly. Finally, we present a cell-scale model of planar polarity based on the vertex model, unifying the themes of this thesis.

Apicobasal polarity is essential for epithelia formation and maintenance. Cell junctions, namely the zonula adherens in Drosophila melanogaster, are the morphological landmarks that define and distinguish the apical from the basal surface. This resulting compartmentalisation is key for the cell and consequently the epithelia. To maintain proper junctions, cells make use of several protein complexes and their interactions. Among these complexes, the Crumbs (Crb) network stands out. Mutations in Crumbs (crb11A22) lead to zonula adherens collapse, consequent loss of apical surface and disaggregation of the epithelia. However, the mechanisms behind this are not known and havenʼt been addressed using modern techniques such as live imaging. Several things came out of the dataset obtained from the FRAP experiments. Firstly, protein kinetics are better described when a double exponential fit curve is used, which raises the possibility that two cell processes might be involved in the recovery observed for the different markers. Another finding was the fact that the kinetics of some polarised protein markers is not the same in every region of the embryo. Distinct areas of the embryo with different morphogenetic activity levels show different kinetics for the same compartment marker. That was the case with SpiderGFP (whole plasma membrane marker) and SASVenus (apical plasma membrane marker) where τ2 was lower in the posterior region of the embryo which is characterised by intense cell movements resulting from convergence extension. DE-CadGFP (zonula adherens marker) and lacGFP (basolateral marker) behaved similarly in the whole embryo. This indicates that convergence extension shows different trafficking needs for the apical surface. In crb11A22, SpiderGFP kinetic spatial differences were not observed. τ2 in the anterior (low level of morphogenesis) is affected and similar to wild type τ2 levels in the posterior. This could pinpoint the fact that the epithelia disaggregation is a result of trafficking failure of apical components. Live imaging of DE-CadGFP in crb11A22 background revealed initial disaggregation in the anterior part of the embryo, which strengthens the idea that Crb is required for adherens junction stabilisation and maintenance.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23571号 / 医博第4785号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 大森 孝一, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The focus of this study was to elucidate the molecular and cellular mechanisms whereby fibroblast growth factors (FGFs) mediate outgrowth of the limb. Specifically, we examined the epistatic relationship between FGF and Wnt/Planar cell polarity (PCP) signaling in establishing cell polarity as a mechanism for outgrowth. By implanting beads into embryonic limbs and lateral plate mesoderm, we established that FGF activates Wnt5a in a gradient fashion. Once it was established that Wnt5a was expressed at the right time and place to turn on PCP signaling, we investigated the ability of Wnt5a to influence cell migration and/or cell polarity. Our analysis revealed that there was no difference in cell migration when cells were exposed to an exogenous Wnt5a source. However, this did not rule out the possibility that cells were responding in a more mild fashion and polarizing toward a Wnt5a source. Live cell imaging was performed to observe the movement and morphology of limb mesenchyme cell cultures in the presence or absence of a Wnt5a expressing cell bolus. It appears as though the cells orient and move in a random fashion regardless of Wnt5a. However, this in vitro method may not truly recapitulate in vivo events. Future studies aim to develop better methods of observing cell polarization in vitro, including developing better methods to tract the movement of cells and observe “PCP” events. Due to the lack of information gathered from our in vitro studies, an in vivo study was conducted to test if FGF is necessary to polarize limb mesenchyme cells. If FGF is turning on Wnt5a and Wnt/PCP signaling is directing cell polarization, then FGF mutant clones will not migrate toward the AER. Therefore, it is expected that these mutant clones would be unable to undergo directed cell movement and/or cell divisions. Early clonal analysis indicates that a response to FGFs appears to be necessary to direct polarized outgrowth of limb mesenchyme.

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