Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11 th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from treatment between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-ulnar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.

Magister Chirurgiae Dentium (MChD) / The author is interested in the morphogenesis of the oral region including the nasopalatine complex. With the intention of undertaking a study of the· embryological development in this area, perusal of available literature failed to reveal a single compreh.ensive description of the reception and handling of embryonic and foetal material, mensuration and preparation for miscroscopy. Human material for embryological study is relatively scarce in· the Republic of South Africa. According to the literature there is, however, a distinct similarity between human and domestic pig development in certain regions, notably the palate. Furthermore, pig embryos and foetuses are available in comparative abundance from sows slaughtered at abattoirs. As a consequence of the above-mentioned factors it was,decided to undertake a -preparatory study in order to firstly evaluate existing methods of handling of embryonic and foetal material and secondly, to statistically evaluate data relating to mass and measurements.'· The aim was to draw a comparison with existing information and to select a sample for investigation. Embryos and foetuses were removed from slaughtered sows in a fresh state and removed to the laboratory immersed in 10 per cent neutral buffered formol saline. In the laboratory foetal membranes were removed, umbilical cords cut and the specimens weighed. They were then placed in Bouin's solution for final fixation and decalcification. Instruments were designed to measure crown-tailroot length, crown-rump length and dorsal profile length. After one day in Bouin's solution all specimens were measured. In order to determine the accuracy of the weighing and measuring procedures ten fixed specimens were weighed and measured on seven consecutive days. Statistical analysis of this data indicated that crown-rump length was the most accurately determinable linear measurement, judged by both the coefficient of variation and the standard deviation. On this basis crown-rump length was chosen as the criterion for selecting the sample to be studied. Correlation between linear measurements and between linear measurements and mass for the entire series showed a very strong positive relationship between all the parameters indicating that a dimensional relationship was maintained during growth. After measuring, the small specimens were embedded whole while larger embryos and foetuses were decapitated. A method was described for trimming and embedding these heads in such a way that subsequent sectioning would take place in a standardised transverse plane. In larger specimens this procedure had to be delayed until demineralization had taken place. Conclusions based on a consideration of data for the entire population included the following: 1. The mean number of specimens per litter was 6,475. 2. The number of pigs per litter stayed relatively constant throughout the period of gestation. 3. Mass showed a greater intra-litter variation than any of the three linear measurements recorded. 4. Relatively, lengths appeared to vary less in older than in younger Ldtt.ers-, irrespective of litter size

Les plantes croissent de manière continue tout au long de leur vie. Elles sont notamment capablesde produire de nouveaux axes de croissance, ce qui nécessite la mise place d’une zone frontière, induitepar l’expression des facteurs de transcription CUP-SHAPED COTYLEDON 1-3 (CUC). Au cours de mathèse, j’ai utilisé les dents formées à la marge des feuilles chez Arabidopsis thaliana comme un modèlepour mieux comprendre le rôle du réseau régulateur centré sur les gènes CUC au cours de lamorphogenèse.La première partie de mon travail a consisté en l’étude des processus en aval de CUC2, le principalrégulateur de la formation des dents. Grâce à l’utilisation d’un système d’expression inductible pour CUC2combiné à des analyses morphométriques et à la quantification de gènes rapporteurs, j’ai montré queCUC2 agit comme un déclencheur primaire et quantitatif de la formation des dents. Plusieurs relaisagissent en aval de CUC2, à des moments et dans des domaines différents, et ensemble permettent à ladent de continuer de croitre.Dans une seconde partie de mon travail, j’ai identifié et caractérisé des régulateurs en amont desgènes CUC. En suivant une approche candidat, j’ai montré que le microARN miR164 et le complexepolycombe PRC2 interagissent et contrôlent finement l’expression de CUC2. De plus, j’ai réalisé un criblesimple hybride en levure suivi d’expériences de validation in planta pour identifier de nouveauxrégulateurs de l’expression des gènes CUC/MIR164. Enfin, j’ai initié une validation fonctionnelle pourcertains de ces nouveaux candidats et montré qu’il s’agit de régulateurs généraux de l’architecture de lapartie aérienne. En décryptant les mécanismes en amont et en aval des gènes CUC, ce travail a permis demettre en évidence de nouveaux aspects de la mise en place des zones frontières et de la manière dont elles régulent l’architecture des plantes. / Throughout their lives, plants are able to produce new axes by differential growth. The formationof such new growth axes depends on the establishment of a boundary domain, which requires the CUPSHAPEDCOTYLEDON 1-3 (CUC) transcription factors. In this work, I used the small outgrowthsformed at the margin of Arabidopsis thaliana leaves as a model to decipher the CUC-centered networkregulating morphogenesis.In the first part of my work, I focused on the events downstream of CUC2, the master regulator ofleaf margin morphogenesis. Using conditional CUC2 expression combined with morphometric analysesand quantification of reporter genes, I showed that CUC2 functions as a primary and quantitative triggerfor morphogenesis. This trigger then acts through multiple relays, which actions spatially and temporallydiffer, and together allow sustained differential growth.In the second part of this work, I identified and characterized upstream regulators of the CUCgenes. In a candidate-based approach, I showed that miR164 and the polycomb complex PRC2 interact totightly control CUC2 expression. Next, I uncovered new potential transcriptional regulators of theCUC/MIR164 genes through a yeast one-hybrid screen followed by an in planta assay. Finally, I initiateda functional study for some of these candidates, which showed that they are general regulators of shootarchitecture. By revealing upstream and downstream components of the CUC-centered network, this workprovides new insights into how boundaries are regulated and how they shape plants.

Les feuilles des plantes sont des organes importants pour la production de biomasse dans la nature car elles sont le siège principal de la photosynthèse, qui permet de transformer la matière minérale en matière organique. Identifier les mécanismes responsables de la morphogenèse, i.e. la genèse de la forme pendant le développement, est donc une question d'intérêt. Pour être analysée, la morphogenèse doit être appréhendée tout au long de la croissance car la forme finale d'une feuille est le résultat de mécanismes coordonnés dans l'espace et le temps. Pour comprendre ce type de processus complexes, la modélisation est une approche de choix. L'objectif de cette thèse était donc de développer des stratégies de quantification et de modélisation de la morphogenèse pour mieux comprendre le développement des feuilles. Pour quantifier la morphogenèse, ma première contribution a été de développer des méthodes pour dater précisément l'apparition des feuilles sur la plante et celle des dentelures sur la marge foliaire, ce qui permet de recaler dans le temps et comparer différentes feuilles en croissance. En calculant les trajectoires de croissance de feuilles moyennes, il est alors possible de préciser où et quand le développement de feuilles peuvent différer, au niveau global ou des dentelures, pendant la croissance. En analysant des feuilles de formes différentes de la plante modèle Arabidopsis thaliana, j'ai ainsi pu montrer que malgré des différences importantes en taille et forme globale, il y a une similarité dans le développement des dentelures. Ces résultats suggèrent qu'il existe des processus identiques qui gouvernent l'apparition et la croissance des dentelures. J'ai ensuite proposé un modèle de développement des feuilles, à partir duquel il est possible de simuler la croissance d'une feuille. Il est basé sur des mécanismes biologiques qui on été identifiés comme étant importants dans la mise en place de la forme. Pour paramétrer le modèle, une approche d'optimisation a été mise au point pour déterminer les paramètres optimaux du modèle. Les résultats obtenus montrent que l'apparition séquentielle des dents ainsi que certains paramètres morphologiques peuvent être bien reproduits par le modèle. / Plant leaves are important for the production of biomass in nature, because they are the main site of photosynthesis, They have various shapes and it has been shown that their morphology influences photosynthesis efficiency. Identifying the mechanisms responsible for morphogenesis, i.e. the genesis of the shape during development, is therefore a matter of interest. To be analyzed, morphogenesis must be apprehended throughout the whole growth because the leaf final form is the result of coordinated mechanisms in space and time. To understand this type of complex processes, modeling is an approach of choice. Consequently, the objective of this thesis was to develop strategies for the quantification and modeling of morphogenesis to better understand leaf development. To quantify morphogenesis, my first contribution was to develop methods to precisely date the appearance of the leaves on the plant, and of the serrations at the leaf margin, allowing to register in time and to compare different growing leaves. Besides, based on mean growth trajectories, it is possible to specify where and when the developments of different leaves differ, at global and serration scales, during growth.By analyzing the development of leaves of the plant model Arabidopsis thaliana that have different shapes, in wild type or in mutants, it has been shown that, despite significant differences in leaf size and shape, there is a similarity in the development of all serrations. These results suggest that there are identical processes that control the appearance and growth of serrations. I proposed two leaf development models, based on biological mechanisms that have been identified, in the literature, as important for the leaf shaping, and also on the quantification of leaf morphogenesis performed in this work. The simulation module, that generates growth trajectories from the model, makes it possible to compare simulated and real developments. To parameterize the model, an optimization approach has been proposed to determine optimal parameters, which minimizes the differences between simulation and real growths. The results showed that the sequential appearance of the teeth as well as important morphological characteristics can be well reproduced by the models.

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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