La morphogénèse des ovocoques, dont fait partie le pathogène humain Streptococcus pneumoniae, implique des processus d’élongation et de division associés à la synthèse de la paroi bactérienne. Le composant majeur de cette paroi est le peptidoglycane, un polymère de sucre réticulé par des chaines peptidiques, qui confère la forme de la bactérie et est essentiel à sa survie. La synthèse de peptidoglycane nécessaire à l’élongation et la division bactérienne est effectuée par des complexes protéiques appelés respectivement « élongasome » et « divisome ». Les mécanismes d’assemblage et l’activité de ces complexes dans la cellule bactérienne restent encore non élucidés. Pour imager l’activité des complexes de synthèse du peptidoglycane in vivo à l’échelle du nanomètre, j’ai développé une méthode faisant appel à des dérivés de D-amino acides, à la chimie click et à la microscopie de localisation de molécules uniques (dSTORM ou direct Stochastic reconstruction microscopy). Cette méthode a permis d’obtenir des images à une résolution d’environ 20 nm, révélant des aspects inattendus de la synthèse du peptidoglycane et remettant en question le rôle de certaines protéines dans la morphogenèse du pneumocoque. En combinant ces observations avec les données de la littérature, un modèle simplifié de la morphogénèse des ovocoques est proposé. / The morphogenesis of ovovcocci, which include the human pathogen Streptococcus pneumoniae, involves elongation and division processes associated with cell wall synthesis. The main component of the cell wall is the peptidoglycan, a polymer made of glycan chains cross-linked by peptide chains, which confers the bacterial shape and is essential for cell survival. Peptidoglycan synthesis required for cell elongation and division is performed by large protein complexes called “elongasome” and “divisome”, respectively. The assembly mechanisms and activity of these complexes in the bacterial cell remain mysterious. To image the activity of the peptidoglycan synthesis complexes in vivo at the nanoscale, I developed a method combining D-amino acid derivatives, click chemistry and single-molecule localization microscopy (dSTORM or direct Stochastic reconstruction microscopy). This method allowed obtaining images at a resolution of about 20 nm resolution, revealing unexpected features of peptidoglycan synthesis and challenging the role of some proteins in pneumococcus morphogenesis. By combining these observations with data from the literature, a simplified model of ovococci morphogenesis is proposed.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20076号 / 医博第4169号 / 新制||医||1018(附属図書館) / 33192 / 京都大学大学院医学研究科医学専攻 / (主査)教授 影山 龍一郎, 教授 斎藤 通紀, 教授 高橋 淳 / 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20561号 / 医博第4246号 / 新制||医||1022(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 秀一, 教授 瀬原 淳子, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The overall architecture and cell types are highly conserved from mammals to teleost fish. The rapid transparent ex utero development in zebrafish allows direct access and precise visualization of all the major events in cerebellar development. The superficial position of the cerebellar primoridum and cerebellum further facilitates in vivo imaging of cerebellar structures and developmental events at cell resolution. Furthermore, zebrafish model have a comprehensive genetic toolbox that allow forward and reverse genetic approaches to study and manipulate gene function. Consequently, zebrafish is emerging as an excellent vertebrate model for studies of molecular, cellular and physiological mechanisms involved in cerebellar development and function at gene, cell and circuit level.

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Observing and studying how life forms behave, i.e., their movement, adaptability, and so on, have enabled human beings to develop new technologies or optimize existing ones. One of the more noticeable phenomena in Nature is morphogenesis. Morphogenetic processes exist in different stages of biological development, from cellular division to tissue and organ formation. It is easy to observe shape development during mophogenesis due to emerging imaging techniques. However, it is hard to understand this process due to its complex organization, and the morphogenetic responses can be induced by a variety of chemicals or mechanical stresses and are subject to the stochastic fluctuation of the environment, making it even more difficult to acquire a fundamental understanding. It is natural to think of mimicking the complex biological process using simplified synthetic approaches. Endowing synthetic protocells with the ability to control their shape and motion autonomously would enable them to perform more like a biological system, but with less complexity. Creating synthetic morphogenesis would potentially further our understanding in biological morphogenetic processes. Moreover, we can borrow some of the morphogenetic functions in engineered materials to achieve a variety of applications, including artificial tissues, self-healing materials, controlled drug delivery, manipulation of soft robots, among others. In this dissertation, we used a synthetic cellular system controlled by a reaction regulated network that imitates the genetic control as a minimal model to understand the potential mechanisms of morphogenesis. Different simulation methods were used depending on the length scales of interest in each problem. We studied the following aspects of the minimal model system: (a) catalytic reaction induced local morphological control of amphiphilic diblock copolymer vesicles; (b) non-equilibrium control over the self-assembled structures of amphiphilic surfactants; and (c) diffusiophoretic/self-diffusiophoretic motion of colloidal particles in response to the concentration gradient field. The results obtained in this thesis work will provide a valuable road-map to guide future experiments.

Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

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.

Epithelial morphogenesis is contingent upon cell shape changes. Cell shape changes are the driving force for the metamorphosis of the adult Drosophila leg from the leg imaginal disc precursor. Genetic analysis has identified several Drosophila genes involved in regulating cell shape changes during leg disc morphogenesis. These include members of the RhoA signaling pathway and the product of the Stubble-stubbloid (Sb-sbd) locus, a transmembrane serine protease. Mutations in the Sb-sbd gene interact genetically with the members of the RhoA signaling pathway, however the nature of the relationship between Sb-sbd serine protease activity and RhoA signaling is not understood. To identify additional components of the RhoA signaling pathway that may help us to understand the role of the Sb-sbd protease in RhoA signaling the Drosophila genome was systematically scanned for genes that interact with Sb-sbd and RhoA mutations using deletions/deficiencies of specified regions of each chromosome. A total of 201 deficiencies uncovering approximately 84.9-91% of the euchromatic genome and spanning the X, second, and third chromosoms were tested. Of the 201 deficiencies tested, five putative interacting genetic regions and one gene within these deficiencies were identified. The candidate gene Eip78C encodes a nuclear steroid hormone receptor previously identified as having an important role in metamorphosis.