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Developmental Evolution of the Optic Region in the Cavefish Astyanax mexicanus / Évolution développementale de la région optique chez le poisson cavernicole, Astyanax mexicanusDevos, Lucie 04 July 2018 (has links)
L’espèce Astyanax mexicanus est composée de deux morphotypes de poissons radicalement différents : le très classique poisson de surface vivant dans des rivières et le poisson cavernicole (CF, cavefish) aveugle et dépigmenté. Ces deux morphotypes diffèrent sur de nombreux aspects, aussi bien en termes de modalités sensorielles, qu’en termes de physiologie ou de comportement. L’approche « Evo-Devo » consiste à tenter de relier des différences développementales précoces à des modifications phénotypiques plus tardives. Dans le cadre de ce travail, nous nous sommes concentrés sur les modifications précoces de l’hypothalamus et de l’œil du CF. Nous montrons que des modifications précoces de signalisation de morphogènes tels que Shh ou Fgf conduisent à une modification de la taille des groupes de neurones peptidergiques au sein de l’hypothalamus, via les facteurs de transcription Lhx, impliqués dans la spécification neuronale. Plus particulièrement, nous montrons l’augmentation de taille des groupes de neurones NPY ainsi qu’hypocretine, qui à son tour provoque une réduction du sommeil chez le CF.Nous nous sommes aussi intéressés à l’oeil du CF, qui commence par se développer avant de dégénérer. Une réduction du quadrant ventral de la rétine avait été précédemment décrit. Nous rafinons cette description grâce à une étude de la régionalisation de la coupe optique du CF qui suggère une réduction de la rétine temporale plus spécifiquement. Nous proposons également une première description de la morphogénèse de l’oeil du CF grace à l’imagerie live de lignées transgéniques fluorescentes. Cette étude révèle un défaut d’invagination de la coupe optique chez le CF. Globalement, ce travail ouvre la voie vers une meilleure compréhension de l’évolution de la tête du CF. / Astyanax mexicanus is a fish species comprising two strikingly different morphotypes : the classical river-dwelling surface fish and the blind depigmented cavefish. These two morphs differ in many aspects in terms of sensorial modalities, physiology and behaviour. In the Evo-Devo approach, we try to link early developmental differences to later phenotypic modifications. Here we focus on the early modification of the hypothalamus and the eye of the cavefish. We show that early signalling modification of morphogens such as Shh or Fgfs lead to the modification of neuropeptidergic clusters in the hypothalamus via the neuronal fate-specifying transcription factors Lhx. More particularly, we show an increase in NPY and hypocretin cluster size. In turn, this increased hypocretin cluster size triggers a reduction of sleep in the cavefish larva.We also examine the embryonic eye of the cavefish which first develops before degenerating. This eye was previously reported to have a reduced ventral retina. We refine this description by studying the regionalisation of the cavefish optic cup and suggest that this reduction concerns more specifically the temporal retina. We also attempt a first description of the cavefish eye morphogenesis by live imaging on fluorescent transgenic lines. This description reveals a defect in the optic cup invagination of the cavefish. Overall, this work started deciphering the developmental evolution of the cavefish head.
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Spatio-temporal and quantitative control of Rho1 activity by GPCR signaling during tissue morphogenesis / Contrôle spatio-temporel et quantitatif de l'activité Rho1 par une signalisation GPCRGarcia De Las Bayonas, Alain 14 December 2018 (has links)
La constriction apicale des cellules du mésoderme et l'intercalation des cellules de l'ectoderme sont contrôlées par des réseaux contractiles d'acto-myosine dans l'embryon de Drosophile. Le niveau d'activation et la polarisation du cytosquelette d'acto-myosine détermine la nature des déformations cellulaires observées. Nous montrons que le GPCR Smog et les protéines G (Gα,Gβγ) en aval, activent la signalisation Rho1 et donc la Myosine-II dans les deux tissus. Dans l'ectoderme, Gα12/13 active Rho1 à la membrane apicale (aussi appelé compartiment médio-apical) tandis que les sous-unités Gβ13F-Gγ1 activent Rho1 en médio-apical et aux jonctions cellulaires. Les mécanismes contrôlant l’activation polarisée de Rho1 dans ce tissu demeurent incompris. Nous montrons ici que deux RhoGEFs, RhoGEF2 et une nouvelle RhoGEF Wireless/p114RhoGEF, activent Rho1 sous le contrôle des protéines G dans l’ectoderme. RhoGEF2 stimule Rho1 en médio-apical sous la dépendance de Gα12/13 alors que Wireless/p114RhoGEF contrôle l’activité de Rho1 aux jonctions avec Gβ13F-Gγ1. RhoGEF2 est présente aux jonctions et en médio-apical tandis que Wireless/p114RhoGEF est uniquement jonctionnelle où elle est recrutée par Gβ13F-Gγ1. Pour finir, Wireless/p114RhoGEF est absente des jonctions dans les cellules du mésoderme. En résumé, des GPCRs contrôlent l’activité spatio-temporelle de Rho1 au moyen de deux modules régulatoires dans l’ectoderme. Les protéines G transduisent le signal en recrutant et en activant deux RhoGEFs complémentaires en médio-apical et aux jonctions. Une variation dans la nature des GPCRs, protéines G ou des RhoGEFs détermine le contrôle tissu-spécifique de Rho1 au cours de la morphogenèse. / Cell apical constriction in the mesoderm and cell intercalation in the ectoderm are controlled by contractile actomyosin networks in the developing Drosophila embryo. The extent of both actomyosin activation and polarization determines the nature of these cell deformations. We find that the GPCR Smog and the downstream G proteins (Gα,Gβγ) activate Rho1 signaling and thereby myosin-II in both tissues. In the ectoderm, Gα12/13 activates Rho1 at the apical membrane (also called medial-apical compartment) while Gβ13F-Gγ1 subunits promote Rho1 activity at the apical membrane and at cell junctions. How such a polarized activation of Rho1 is achieved remains unclear. Here, we show that two RhoGEFs, RhoGEF2 and a previously uncharacterized RhoGEF Wireless/p114RhoGEF, control Rho1 activity downstream of G proteins in the ectoderm. RhoGEF2 activates medial-apical Rho1 under control of Gα12/13 and Wireless/p114RhoGEF is required to mediate Gβ13F-Gγ1-dependent activation of Rho1 at junctions. RhoGEF2 is present both at junctions and at the apical membrane. In contrast, Wireless/p114RhoGEF only localizes at junctions together with Gβ13F-Gγ1 which recruit the GEF. Finally, we show that Wireless/p114RhoGEF is absent from junctions in the mesoderm. Collectively, GPCRs shape Rho1 activity through distinct biochemical modules in the ectoderm. Heterotrimeric G proteins transduce the signal by recruiting and activating two complementary RhoGEFs apically and at junctions. Variation in type of GPCRs, G proteins or RhoGEFs underlie the tissue-specific control of Rho1 signaling during morphogenesis.
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Physical Aspects of Local Solid Tumor GrowthKubitschke, Hans 05 June 2020 (has links)
Krebszellen haben gemeinsame Eigenschaften, wie unbegrenztes Wachstumspotential und die Vermeidung von Apoptose. Krebs kann als systemische Erkrankung angesehen werden und es reicht daher nicht aus, molekulare Details von Krebs zu verstehen, sondern auch emergente physikalische Eigenschaften von Krebs auf mehreren Größenskalen von Genen über Zellen bis hin zu Geweben.
Diese Arbeit konzentriert sich auf physikalische Eigenschaften die an der Krebsprogression, der Migration von Krebszellen und dem Krebswachstum beteiligt sind. Die Migration von Krebszellen führt zur Fähigkeit zur Metastasierung, der häufigsten Ursache für krebsbedingten Tod. Der Schlüssel zu diesem Prozess ist die Verformbarkeit von Krebszellen beim Durchqueren der dichten Mikroumgebung aus extrazellulärer Matrix und anderen Zellen. Der genaue Beitrag des Aktin- und Mikrotubuli-Netzwerks zur zellulären elastischen Verformung und Entspannung ist wichtig und wurde untersucht. Ein wichtiges Ergebnis ist, dass bei kleinen Verformungen (<5%) die Aktin-Filamente die viskoelastische Zellverformung unter mechanischer Belastung dominieren und Mikrotubuli die Zellrelaxation bestimmen, während bei größeren Verformungen (>5%) Aktin-Filamente und Mikrotubuli gleichermaßen zur Zellverformung und -relaxation beitragen. So sind die Mikrotubuli für die Migration in Mikroumgebungen von größerer Bedeutung, als es die aktuelle Literatur vermuten lässt.
Ein initial gebildeter bösartiger Tumor tritt typischerweise in eine Wachstumsphase ein, in der das umgebende Gewebe verdrängt und eingedrungen wird. Für ein optimales klinisches Behandlungsergebnis sollte der Primärtumor so gut wie möglich entfernt werden, was die genaue Erkennung der Tumorfront und die Identifizierung der Gewebe mit dem Risiko einer Krebsinfiltration beinhaltet. In dieser Arbeit werden natürliche Hindernisse und Grenzen für das Krebswachstum, wie z.B. Fasziengewebsgrenzen oder Gewebekompartimentgrenzen, basierend auf klinischen Daten von Gebärmutterhalskrebs analysiert, die aus der pathologischen Untersuchung von chirurgisch resezierten Tumoren von 518 Patienten gewonnen wurden. Die Wachstumsgrenzen wurden als embryonale Gewebeentwicklungsgrenzen identifiziert und betonen, dass Krebs Entwicklungsmerkmale aufweist, die häufig in der Embryogenese vorkommen. Das gefundene Tumorwachstumsmuster und die -form widersprechen dem das das vorherrschende Dogma der isotropen Tumorwachstum, welches der chirurgischen Tumorresektion und Strahlentherapie zugrunde liegt. Die Tumorform-Distribution weist starke Abweichungen von sphärischer Symmetrie auf, was darauf hindeutet, dass Tumore durch entwicklungsbiologische Kompartimente und deren Kompartimentsgrenzen begrenzt und geformt werden. Computersimulationen liefern auch den Nachweis, dass die klinisch gefundene Tumorinfiltrationswahrscheinlichkeit von Geweben nicht auf der metrischen Entfernung des gefährdeten Gewebes zum Gewebe der Tumorherkunft basiert, sondern auf der ontogenetischen Verwandtschaft der Gewebe.
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Continuum mechanics of developing epithelia:: Shaping a fly wingPopovic, Marko 24 May 2017 (has links)
Developing tissues are out-of-equilibrium systems that grow and reshape to form organs in adult animals. They are typically composed of a large number of cells. The constitutive cells of a tissue perform different roles in tissue development and contribute to the overall tissue shape changes.
In this thesis, we construct a hydrodynamic theory of developing epithelial tissues. We use it to investigate the developing wing of the fruit fly Drosophila melanogaster. This theory relates the coarse-grained cell scale properties to the large-scale tissue flows. We explicitly account for the active cellular processes in the tissue that drive tissue flows. In our description of the tissue, we also include the memory effects that are necessary to account for the experimental observations. These memory effects have a significant influence on the tissue rheology.
Using this hydrodynamic theory we analyze shear flow in a developing fruit fly wing tissue. We find that the active cellular processes contribute to overall tissue flows and that memory effects are present in the wing tissue. We investigate consequences of these findings on the rheology of tissue shear flow. We find that the memory effects give rise to an inertial response that leads to oscillations in the tissue but it does not stem from the wing mass. Finally, we describe how the tissue rheology is affected by different boundary conditions.
We then investigate the area changes during the pupal wing development and we construct a mechanosensitive model for the cell extrusion rate in the pupal wing. Analysis of cell extrusions in the context of this model also allows us to extract information about the cell division properties.
Boundary connections between the wing tissue and surrounding cuticle are crucial for the proper development of the pupal wing. A dumpy mutant wing is strongly misshaped during the pupal wing morphogenesis. We use a simple model for the wing to show that the dumpy mutant wing can be described as a wild type wing with compromised boundary conditions.
Finally, we analyze cell properties and tissue flows in a developing wing disc epithelium. Motivated by the observation of radially oriented active T1 transitions in the wing disc epithelium, we use the hydrodynamic theory to investigate the influence of such T1 transitions on stresses in the tissue. We show that sufficiently strong radially oriented active T1 transitions can contribute to the control of the tissue size.
Results obtained in this thesis extend our understanding of the fly wing tissue rheology and the role of internal and external forces in the proper shaping of the wing epithelium. The hydrodynamic theory we use to describe the fly wing development provides a set of phenomenological parameters that characterize the tissue mechanics and can be experimentally measured. Therefore, we expect that future research will include and extend the hydrodynamic theory presented in this thesis.
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Molecular, Cellular and Mechanical basis of Epithelial Morphogenesis during Tribolium EmbryogenesisJain, Akanksha 11 September 2018 (has links)
Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events.
During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds.
In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the
main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos.
In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions.
Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page
List of Tables v
List of Figures vii
1 Introduction 1
1.1 Evo-Devo of insects 3
1.2 Tribolium castaneum 5
1.3 Fluorescence live imaging and lightsheet microscopy 10
1.4 Morphogenesis 15
1.5 Thesis objective 29
2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33
2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35
2.2 Double labelling of Tribolium embryos 37
2.3 Image processing with Fiji 37
2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44
2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47
2.6 Summary 59
3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61
3.1 Tc-Squash dynamics during Tribolium embryogenesis 64
3.2 Myosin drives basal cell closure during blastoderm cellularisation 66
3.3 Myosin shows planar polarity in the embryonic tissue 69
3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71
3.5 Myosin pulses during apical constriction of mesoderm cells 74
3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77
3.7 Summary 77
4 A supracellular actomyosin cable operates during serosa epiboly 79
4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81
4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82
4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87
4.4 Serosa cells increase in area till circular serosa window stage 89
4.5 Tension in the serosa tissue increases during epibolic expansion 89
4.6 Serosa cells decrease their apical areas after laser ablation 92
4.7 Tension in the actomyosin cable increases during serosa epiboly 93
4.8 Myosin dynamics at the cable changes between early and serosa window stage 96
4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98
4.10 Myosin dynamics at the cable during serosa window closure 101
4.11 Tension in the cable is not relieved after multiple laser cuts 103
4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105
4.13 Summary 109
5 Discussion 111
5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111
5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114
5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119
6 Materials and Methods 123
6.1 Tribolium stock maintenance 123
6.2 RNA extraction and cDNA synthesis 124
6.3 Cloning of templates for mRNA synthesis and transgenesis 124
6.4 dsRNA synthesis for RNAi experiments 126
6.5 Capped, single stranded RNA synthesis 126
6.6 Fluorescence image acquisition 27
A Appendix 131
Bibliography 143
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Evoluční a teoreticko-biologické aspekty díla Bohumila Němce / Evolutionary and theoretical aspects of the biological work of Bohumil NěmecLoginov, Ivan January 2020 (has links)
Plant physiologist Bohumil Němec is one of the most important people in the history of Czech science. In this thesis, crucial concepts and theories that B. Němec developed in his scientific and popularizing publications are described and put in context. In addition, materials from the personal fond of B. Němec located in the Archive of the CAS were used. Particular questions tackled in this thesis are those of stimuli transmission in plants, the starch-statolith theory of gravitropism, morphaesthesia, organogenes, Němec's approach to evolutionary theory, and genetics, and the division of organisms in two groups. Moreover, Němec's role in the contemporary debates about vitalism, mechanism, and the effect of these two philosophies on the approach towards scientific research is indicated. By that, Němec's transition from the mechanism to the so-called physiology of stimuli (Rheizphysiologie) is illustrated. The outcome of the thesis is broadening the knowledge in the history of plant physiology, especially the history of the natural sciences in the Czech context.
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Production of Congenital Limb Defects With Retinoic Acid: Phenomenological Evidence of Progressive Differentiation During Limb MorphogenesisKwasigroch, Thomas E., Kochhar, D. M. 01 November 1980 (has links)
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.
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The handling of undated pig embryos and foetuses as a prelude to histological studies of morphogenesis in the oral regionvan Rensburg, Barend. Gabriel January 1976 (has links)
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
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Towards the elucidation of the CUP-SHAPED COTYLEDON-centered network duringArabidopsis thaliana leaf development / Vers une meilleure compréhension du réseau de régulation centré sur CUP-SHAPEDCOTYLEDON au cours du développement de la feuille d’Arabidopsis thalianaMaugarny-Calès, Aude 10 November 2017 (has links)
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.
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Quantification et modélisation de la morphogenèse foliaire / Quantification and modeling of leaf morphogenesisOughou, Mohamed Said 22 March 2019 (has links)
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.
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