京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21641号 / 医博第4447号 / 新制||医||1034(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 河本 宏, 教授 三森 経世, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21673号 / 医博第4479号 / 新制||医||1036(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 松田 道行, 教授 滝田 順子, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Monitoring, tracking, and analyzing the dynamic growth of a living organism is essential to understanding its response to changes in its surrounding environment. Imaging tools to study these dynamics at spatial and temporal scales with optimal resolution rely on high-performance instrumentations. These systems are generally costly, stationary, and not flexible. In addition, performing non-destructive high-throughput phenotyping to extract roots' structural and morphological features remains challenging. We developed the MultipleXLab: a modular, mobile, and cost-effective robotic root imager to tackle these limitations. Among its advantages associated with a large field-of-view, integrated programmable plant-growth lighting, and high magnification with a high resolving power, the system is useful for a wide range of biological applications. We have also created the MultipleXLab Advanced; this configuration turns the system into a mobile environmental chamber by also featuring temperature control and automated irrigation. Another system we developed was the MultipleXLab Advanced Fluorescence to allow fluorescence imaging with a resolution that competes with a fluorescence binocular or even a fluorescence microscope. Furthermore, we have implemented various technologies and techniques to facilitate 3D imaging and quantification, ranging from X-ray micro-Computed Tomography to 3D segmentation of tissues, cells, and cellular compartments within the cell imaged using Confocal Laser Scanning Microscopy. For future research, we have conceptualized an upscaled system named MultipleXLabXL. This larger system will allow tracking, monitoring, and quantifying root growth of a much higher number of seedlings for more extended periods.

This thesis was done in collaboration with Sorbonne University as part of a double degree Cotutelle. / During development, cell differentiation frequently occurs upon signaling from concentration or activity gradients of molecules called morphogens. These molecules control in a dose-dependent manner the expression of sets of target genes that determine cell identity. A simple paradigm to study morphogens is the Bicoid gradient, which determines antero-posterior patterning in fruit fly embryos. The Bicoid transcription factor allows the rapid step-like expression of its major target gene hunchback, expressed only in the anterior half of the embryo. The general goal of my thesis was to understand how the information contained in the Bicoid morphogen gradient is rapidly interpreted to provide the precise expression pattern of its target. Using the MS2 system to fluorescently tag specific RNA in living embryos, we were able to show that the ongoing transcription process at the hunchback promoter is bursty and likely functions according to a two-state model. At each nuclear interphase, transcription is first observed in the anterior and it rapidly spreads towards the posterior, as expected for a Bicoid dose-dependent activation process. Surprisingly, it takes only 3 minutes from the first hints of transcription at the anterior to reach steady state with the setting of a sharp expression border in the middle of the embryo. Using modeling taking into account this very fast dynamics, we show that the presence of only 6 Bicoid binding sites (known number of sites in the hunchback promoter) in the promoter, is not sufficient to explain the establishment of a sharp expression border in such a short time. Thus, either more Bicoid binding sites or inputs from other transcription factors could help reconcile the model to the data. To better understand the role of transcription factors other than Bicoid in this process, I used a two-pronged strategy involving synthetic MS2 reporters combined with the analysis of the hunchback MS2 reporter in various mutant backgrounds. I show that the pioneer factor Zelda and the Hunchback protein itself are also critical for hunchback expression, maternal Hunchback acting at nuclear cycle 11-12, while zygotic Hunchback is acting later at nuclear cycle 13-14. The synthetic reporter approach indicate that in contrast to Hunchback and Caudal, Bicoid is able to activate transcription on its own when bound to the promoter. However, the presence of 6 Bicoid binding sites only leads to stochastic activation of the target loci. Interestingly, the binding of Hunchback to the Bicoid-dependent promoter reduces this stochasticity while Caudal might act as a posterior repressor gradient. Confronting these experimental data to theoretical models is ongoing and should allow to better understand the role of transcription factors, other than Bicoid, in hunchback expression at the mechanistic level. / Thesis / Doctor of Philosophy (PhD) / Have you ever wondered how a single cell can become a full grown organism? Well it starts when an egg and sperm fuse together. As time passes this single cell divides over and over again until an organism is formed. During this developmental process, somehow the cells know exactly where they are and what they need to become so that they form the organism. However, we don’t fully understand this process and this is what we hope to answer with our research: How do the cells know where they are and what they need to become during development? We study this process in the fruit fly. Although fruit flies might not look a lot like us, during early embryonic development we are quite similar, so we can try to answer these questions in fruit flies and what we find might be relevant to other organisms like us. During development, the first element that an embryo needs to know is the orientation of its body, where the head and tail, the left and right and the back and front of the body will be. We concentrate on studying how the head to tail axis, which we call the anterior-posterior axis, is formed. To know where the head is going to be, the embryo releases proteins called morphogens that broadcast instructions to other genes so that cells know where they are and what they should become. We study a morphogen called Bicoid. Its concentration is high in the anterior, the region that will become the head of the embryo, and lower as you move towards the posterior where the tail will form. Bicoid activates a gene called hunchback, which ends up dividing the embryo in two large parts, the top and the bottom. However, Bicoid’s message fades away during each cell division and needs to be read again at the beginning of each new nuclear cycle. So how is the message read and how long does this process take? This last question is particularly critical during the period of very fast cell division. My thesis tries to answer this question. We found out that it takes 3 minutes for a nuclei to read the Bicoid concentration, activate hunchback and express it correctly. However, in contrast to what was believed before, or namely, that only Bicoid was involved in this process, we found out that other players are involved in helping relay this message. This way hunchback can accurately divide the body in two parts exactly in the middle and without mistake in such a short period of time.

One of the best-known structures in Drosophila is the sex comb, an arrangement of modified bristles on the tarsal forelegs of males. This complex, sexually-dimorphic trait shows striking variation among closely related species, although most other aspects of the tarsal bristle pattern have been conserved. I studied the development of the sex comb in the model organism Drosophila melanogaster and six related species. I confirmed that the D. melanogaster sex comb, although longitudinal in the adult, originates in a transverse orientation and rotates during development, and showed that this process occurs through male-specific convergent extension. However, in the species that I examined that have longitudinally-oriented sex combs that extend the full length of the tarsus, including D. ficusphila and two species of the montium subgroup, the sex comb does not rotate, and instead forms from two longitudinal rows that converge during development. Another species of the montium subgroup, D. nikananu, has a sex comb that is convergently similar to D. melanogaster, but forms in a manner typical of its subgroup, showing that very similar combs can be formed through different processes. In all species, there is a strong correlation between the position of the sex comb and the transverse bristle row on the foreleg tarsus just proximal to it. To test whether it is possible to violate this apparent constraint on development, I perturbed the expression of the leg patterning gene dachshund to generate ectopic sex combs in D. melanogaster. I found that while most patterns showed the same correlation, a few circumvent the constraint. I also demonstrated that the ectopic combs were formed non-autonomously and that overexpression of dachshund can transform certain aspects of the sex comb phenotype to resemble the transverse bristles to which they are homologous.

One of the best-known structures in Drosophila is the sex comb, an arrangement of modified bristles on the tarsal forelegs of males. This complex, sexually-dimorphic trait shows striking variation among closely related species, although most other aspects of the tarsal bristle pattern have been conserved. I studied the development of the sex comb in the model organism Drosophila melanogaster and six related species. I confirmed that the D. melanogaster sex comb, although longitudinal in the adult, originates in a transverse orientation and rotates during development, and showed that this process occurs through male-specific convergent extension. However, in the species that I examined that have longitudinally-oriented sex combs that extend the full length of the tarsus, including D. ficusphila and two species of the montium subgroup, the sex comb does not rotate, and instead forms from two longitudinal rows that converge during development. Another species of the montium subgroup, D. nikananu, has a sex comb that is convergently similar to D. melanogaster, but forms in a manner typical of its subgroup, showing that very similar combs can be formed through different processes. In all species, there is a strong correlation between the position of the sex comb and the transverse bristle row on the foreleg tarsus just proximal to it. To test whether it is possible to violate this apparent constraint on development, I perturbed the expression of the leg patterning gene dachshund to generate ectopic sex combs in D. melanogaster. I found that while most patterns showed the same correlation, a few circumvent the constraint. I also demonstrated that the ectopic combs were formed non-autonomously and that overexpression of dachshund can transform certain aspects of the sex comb phenotype to resemble the transverse bristles to which they are homologous.

<p>The gonad is a unique primordial organ that retains the ability to adopt one of two morphological fates through much of mammalian embryonic development. Previous work in our lab found that dimorphic vascular remodeling was one of the earliest steps during sex-specific morphogenesis. In particular, vessels in XY gonads display highly ordered behavior that coincides with testis cord formation. It was unknown how the vasculature may influence testis cord morphogenesis and, if so, how this was mechanistically related to sex determination. The work in this thesis addresses a single over-arching hypothesis: Male-specific vascular remodeling is required for testis morphogenesis and orchestrates differentiation of the XY gonad. </p><p>To address this question we have modified and developed techniques that allow us to isolate aspects of vascular behavior, gene expression, and endothelial influence on surrounding cells. In particular, the application of live imaging was instrumental to understanding the behavior of various gonadal cell-types in relation to remodeling vessels. It is difficult to grasp the complexity of an organ without understanding the dynamics of its constituents. A critical aim of my work was to identify specific inhibitors of the vasculature that do not affect the early stages of sex determination. Combining inhibitors, live imaging, cell sorting, qRT-PCR, mouse models, and whole organ culture has led to a far richer understanding of how the vasculature behaves and the cell-types that mediate its influence on organ morphogenesis. The beauty of our system is that we do not have to settle for a snapshot of the fate of cells in vivo, but can document their journeys and their acquaintances along the way. </p><p>Vascular migration is required for testis cord morphogenesis. Specific inhibitors revealed that in the absence of vessels, testis cords do not form. The work below shows that vessels establish a feedback loop with mesenchymal cells that results in both endothelial migration and subsequent mesenchymal proliferation. Interstitial control of testis morphogenesis is a new model within the field. The mechanisms regulating this process include Vegf mediated vascular remodeling, Pdgf induced proliferation, and Wnt repression of coordinated endothelial-mesenchymal dynamics. Our work also suggests that vascular patterning underlies testis patterning and, again, is mediated by signals within the interstitial space not within testis cords themselves. </p><p>A final aspect of my work has been focused on how vessels continue to influence morphology of the testis and the fate of surrounding cells. Jennifer Brennan, a graduate student in our lab, previously showed that loss of Pdgfr&#945; antagonizes cord formation and development of male-specific lineages. The mechanisms and cell-types related to this defect were not clear. I began to reanalyze Pdgfr&#945; mutants after finding remarkable similarity to gonads after vascular inhibition. This work is providing data suggesting that vessels are not simply responsible for testis morphology but also for the fate of specialized cells within the testis. On the whole, this thesis describes specific roles for endothelial cells during gonad development and mechanisms by which they are regulated.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22687号 / 医博第4631号 / 新制||医||1045(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 山下 潤, 教授 木村 剛, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Les vésicules synaptiques (VSs) sont essentielles pour la neurotransmission. Les recherches actuelles se focalisent sur la caractérisation de leur contenu en neurotransmetteurs, leur cinétique de libération, leur distribution et leur mobilité. Les VS ne sont pas présentes exclusievement en paquet dans les boutons présynaptiques mais sont echangées de façon dynamique avec le reste de l’axone dans un super-contingent (super-pool). Notre laboratoire a précédement montré que le transporteur vésiculaire de glutamate de type 1 (VGLUT1) jouerait un rôle dans la régulation du super-pool. Mon projet de thèse se focalise sur la mobilité des VS dans les axones. En premier lieu, j’ai généré une souris gain de fonction VGLUT1mEos2 afin d'étudier la mobilité des VSs et de mieux caractériser le super-pool. Ensuite j’ai engagé une étude des relation entre la structure de VGLUT1 et ses fonctions afin d’identifier les signatures moléculaires responsable de la régulation de la taille du super-pool. J’ai identifié le second motif poly-proline à l’extremité C-terminale de VGLUT1 comme étant nécessaire et suffisante pour induire une diminution de la taille du super-pool des VSs. Pour conclure mes travaux de thèse ont contribué à la compréhension du rôle de VGLUT1 dans la régulation de la mobilité des VSs et à fournir les outils nécessaires pour de futures investigations concernant la physiologie du super-pool. / Synaptic vesicles (SVs) are essential for neurotransmission, and more efforts are needed for better understanding their neurotransmitter content, release kinetics, distribution and mobility. SVs are not only clustered in presynaptic boutons, but also dynamically shared among multiple en passant presynaptic boutons, a phenomenon named SV super‐pool. Previous work from our laboratory suggested that the Vesicular GLUtamate Transporter 1 (VGLUT1) may play a role in regulating SV super-­pool size beyond loading glutamate into SV. My Ph.D project is focused on SVs mobility in axons. Firstly, I generated a VGLUT1mEos2 knock-in (KI) mouse line, which provides extended possibilities to study the SV trafficking and characterize SV super‐pool. Secondly, I engaged in a thorough VGLUT1 structure‐function analysis. I identified that VGLUT1 tends to cluster SVs in the presynaptic boutons and reduce SVs exchange with the super‐pool via the second poly‐proline motif of its C­‐terminus. Overall, my Ph.D work contributes to the knowledge of the role of VGLUT1 in regulating SVs mobility and provides new tools for the further investigations on SV super-­pool physiology.