The vertebrate tail is unique for each species and fulfils a broad spectrum of functions. In the axolotl (Ambystoma mexicanum), a tailed amphibian, the tail constitutes one-third of the full body length and is necessary for swimming. Despite its size, most of the tail's tissues are derived from the posterior neural plate of the neurula. Although giving rise to neuronal structures of the central nervous system along most of its length, the most posterior part of the neural plate develops preponderantly into presomitic mesoderm (PSM) which forms muscle, bone and cartilage of the tail and posterior trunk. During development, the posterior neural plate reverses its orientation during an anterior turn movement (Taniguchi et al., 2017). Cells of the most posterior plate region become now localised in an anterior position while previously more anterior neural plate cells land at a more posterior site. Simultaneously, the axial neural tube and notochord extend themselves posteriorly. The PSM, developing bilaterally to the central axis, is integrated into posterior tail expansion while forming new somites at its anterior end. It is still elusive which morphological changes the PSM undergoes to facilitate tail formation and posterior elongation of the embryo. Furthermore, it remains enigmatic in what way PSM cells change their shape, orientation, migration behaviour and distribution to meet the requirements needed for adjusting PSM and somite morphology. With homotopic tissue transplantations of posterior neural plate cells from a gfp-expressing donor to a white (d/d) recipient, enabled specific labelling of all mesodermal cells of the tail. Otherwise, mesodermal cells of the trunk and tail can not be distinguished, neither genetically nor morphologically. With this cell labelling approach, the entire tail mesoderm could be imaged in toto. Thus, measurements of the morphological changes of the PSM and cell tracking in 3D was possible during development. With this technique, posterior neural plate cells could be shown to form parts of the posterior neural tube, the entire posterior PSM and the somites of the tail. During this course of development, the PSM becomes longer but does not increase its volume. Only when forming the somites, an increase in volume could be measured in the mesoderm. Single-cell labelling showed an anterior shift of cell movement led by medial PSM cells and followed by more laterally located cells. The anterior displacement happens simultaneously to the posterior elongation of the embryo. A hypothetical push by newly generated cells at the tail tip could be ruled out. Mitotic cells were evenly distributed in all tissues of the tail with a low proliferation rate. The morphological changes and anterior relocations of the tail mesoderm could, therefore, mainly be explained by cell migration. Therefore, further analyses focussed on cell migration, particularly on cellular characteristics displayed during migration such as shape, orientation, volume, distribution and filopodia organisation to obtain more profound information about how PSM cells migrate and contribute to somite formation. The net movement of tail elongation is directed posteriorly regardless of anteriorly relocating PSM cells. That is only feasible if a lateral expansion of the PSM by laterally migrating PSM cells is counteracted. There have been no studies on the lateral boundary so far. In the axolotl, the PSM is covered laterally by a two-layered epidermis and a fibronectin-rich extracellular matrix. After removing the tail epidermis, operated embryos showed missing or malformed tails, especially with lateral and dorsal curvatures and shortenings. Tail mesoderm examined in these cases showed an increased PSM volume and a lateral expansion of the tissue. A nearly normal tail developed when, after removing the epidermis, the embryos developed in 1% agarose supplemented with fibronectin. In contrast, a simple covering of the PSM with a nitrocellulose membrane, incubation in the softer methylcellulose or in agarose without fibronectin did not rescue tail formation. The lateral pressure on the PSM and a fibronectin-rich extracellular matrix seem necessary to preserve the tissue architecture of the PSM during tail formation. This study unravels the behaviour of individual PSM cells during their morphogenesis from single cells in the posterior plate of the neurula until somite formation in the tail bud. Overall, with specific labelling of tail mesodermal cells, their contribution to PSM morphology could be elucidated, and a more detailed model of tail elongation could be proposed: The posterior expansion of the neural tube and notochord pushes the posterior neural plate tissue posteriorly and squeezes the cells into an elongated mediolaterally oriented form. Labelling experiments of small individual cell groups showed that the ventral posteriormost cells are the first to escape this pressure by relocating anteriorly. Then, more anteriorly located cells follow, as well as dorsally located cells. These movements explain the anterior turn. Thereby, mesodermal cells start to migrate randomly, become elongated and change their orientation from mediolateral to anterior-posterior. Random cell migration leads to homogeneous cell mixing, which results in an aligned uniform tissue of trunk and tail PSM. The lateral constriction by the epidermis channels the undirected migration movements in an anterior direction. In this way, cells are directed towards the site of somite formation, the PSM narrows, and the embryo elongates posteriorly. This extension model includes the individual cell behaviour, which on the whole shapes PSM morphology. The analysed dynamic morphological changes of the PSM can be linked to the developmental processes of the tail and the posterior elongation of the axis.:1 Introduction 1.1 Embryonic tail formation . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1.1 Mechanism of tail formation . . . . . . . . . . . . . . . . . . . . . 1 1.1.2 Molecular determination of cell populations in the tail bud . . . . . 5 1.2 Axial elongation of the vertebrate body plan . . . . . . . . . . . . . . . . . 8 1.2.1 Anterior body elongation (elongation of the trunk) . . . . . . . . . 8 1.2.2 Posterior body elongation (tail elongation) . . . . . . . . . . . . . . 9 1.3 Studying tissue morphology during development . . . . . . . . . . . . 11 1.4 Aim of the project . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 12 2 Materials 2.1 Chemicals and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2 Antibodies and dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3 Techniqual equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3 Methods 3.1 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.1.1 Breeding of axolotls and embryo collection . . . . . . . . . . . . 19 3.1.2 Injections with the vital dye DiI . . . . . . . . . . . . . . . . . . . 19 3.1.3 Tissue transplantation techniques . . . . . . . . . . . . . . . . . . . 19 3.2 Immunohistochemical staining . . . . . . . . . . . . . . . . . . . . . . . . 20 3.2.1 Vibratome sections . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.2.2 Whole-mount staining . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.3 Optical tissue clearing protocols . . . . . . . . . . . . . . . . . . 21 3.3.1 Ethyl cinnamate based optical tissue clearing protocol . . . . . . . 21 3.3.2 SeeDB optical clearing protocol . . . . . . . . . . . . . . . . . . . . 22 3.4 Image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.4.1 3D image generation and processing . . . . . . . . . . .. . . . . . 22 3.4.2 Length measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.4.3 Manual segmentation . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4.4 Automatic segmentation . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5 Determination of cellular parameters . . . . . . . . . . . . . . .. . . . . 25 3.5.1 Cell shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5.2 Cell and tissue volume . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5.3 Cellular distribution . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5.4 Closest neighbour analysis . . . . . . . . . . . . . . . . . . . . . . . 26 3.5.5 Cell orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.5.6 Length and orientation of filopodia . . . . . . . . . . . . . . . . . . 31 3.5.7 Distance of cells to a plane . . . . . . . . . . . . . . . . . . . . . . . 31 3.5.8 Mitotic rate and spindle orientation . . . . . . . . . . . . . . . . . 32 4 Results 4.1 The presomitic mesoderm is associated with axial elongation. . . . . . 33 4.1.1 Elongation of the body axis . . . . . . . . . . . . . . . . . . . . . . 33 4.1.2 Contribution of different tissues . . . . . . . . . . . . . . . . . . . . 34 4.1.3 Differential contribution of mesoderm and epidermis . . . . . . . . . 40 4.1.4 Dual potential of mesodermal progenitors . . . . . . . . . . . . . . . 42 4.1.5 Mesodermal tissue expansion . . . . . . . . . . . . . . . . . . . . . 46 4.2 Cellular behaviour influences mesodermal morphology . . . . . . . . . 50 4.2.1 Cell division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4.2.2 Positional changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.2.3 Cellular characteristics . . . . . . . . . . . . . . . . . . . . . . . . . 59 Cell shape changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Change of cell orientation . . . . . . . . . . . . . . . . . . . . . . . 61 Orientation of filopodia . . . . . . . . . . . . . . . . . . . . . . . . . 63 Cell distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 4.3 The epidermis fascilitates mesodermal tissue integrity . . . . . . .. . . . 67 4.3.1 Mesodermal tissue integrity . . . . . . . . . . . . . . . . . . . . . . 68 4.3.2 Malformed tails after epidermis removal . . . . . . . . . . . . . . . 70 4.3.3 Alteration in mesodermal tissue dimensions . . . . . . . . . . . . . 73 4.3.4 Alteration of cell density after epidermis removal . . . . . . . . . . 77 4.3.5 Rescue of tail formation . . . . . . . . . . . . . . . . . . . . . . . . 80 5 Discussion 5.1 Cell migration of the presomitic mesodermal cells . . . . . . . . .. . . . 85 5.1.1 Continuity of gastrulation movements . . . . . . . . . . . . . . . . . 85 5.1.2 Directed migration . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 5.1.3 Random cell migration . . . . . . . . . . . . . . . . . . . . . . . . . 88 5.1.4 Lateral mechanical constriction . . . . . . . . . . . . . . . . . . . . 90 5.2 Non-volumetric growth of the presomitic mesoderm . . . . . . . . . . . . . 91 5.3 Models of tail presomitic mesoderm formation . . . . . . . . . . . . . . . . 93 / Der Schwanz der Wirbeltiere ist bei jeder Art einzigartig und erfüllt ein breites Spektrum an Funktionen. Beim Salamander Axolotl (Ambystoma mexicanum), macht der Schwanz ein Drittel der gesamten Körperlänge aus und ist zum Schwimmen notwendig. Trotz seiner Größe stammen die meisten Gewebe des Schwanzes von der posterioren Neuralplatte der Neurula ab. Obwohl der größte Teil der Neuralplatte neuronale Strukturen des Zentralnervensystems hervorbringt, entwickelt sich der posteriore Teil der Neuralplatte überwiegend zu präsomitischem Mesoderm (PSM), das Muskeln, Knochen und Knorpel des Schwanzes und des hinteren Rumpfes bildet. Während der Entwicklung kehrt die posteriore Neuralplatte ihre Orientierung in einer anterioren Drehbewegung um (Taniguchi et al., 2017). Zellen der hintersten Plattenregion werden in eine anteriore Position verschoben, während zuvor anteriorere Neuralplattenzellen an einer posterioren Stelle landen. Gleichzeitig verlängert sich das axiale Neuralrohr und das Notochord nach posterior. Das PSM, das sich bilateral zur Zentralachse entwickelt, ist im Prozess der Schwanzverlängerung involviert, während es gleichzeitig an seinem vorderen Ende neue Somiten bildet. Es ist immer noch unklar, welche morphologischen Veränderungen das PSM durchläuft, um die Schwanzbildung und die posteriore Ausdehnung des Embryos zu ermöglichen. Darüber hinaus ist unbekannt, auf welche Weise PSM-Zellen ihre Form, Orientierung, ihr Migrationsverhalten und ihre Verteilung ändern, die für eine Veränderung der PSM- und Somitenmorphologie erforderlich sind. Mit homotopen Gewebetransplantationen von posterioren Neuralplattenzellen von einem gfp-exprimierenden Spender auf einen weißen (d/d) Empfänger, konnte eine spezifische Markierung aller mesodermalen Zellen des Schwanzes erreicht werden. Andernfalls können mesodermale Zellen des Rumpfes und des Schwanzes weder genetisch noch morphologisch unterschieden werden. Mit diesem Zellmarkierungsansatz konnte das gesamte Schwanzmesoderm in toto abgebildet werden. So waren Messungen der morphologischen Veränderungen des PSM und Zellverfolgung in 3D während der Entwicklung möglich. Mit dieser Technik konnte gezeigt werden, dass die Zellen der posterioren Neuralplatte Teile des posterioren Neuralrohrs, das gesamte posteriore PSM und die Somiten des Schwanzes bilden. Dabei wird das PSM länger, ohne sein Volumen zu vergrößern. Erst während der Bildung von Somiten wurde eine Volumenzunahme gemessen Einzelzellmarkierungen zeigten eine anteriore Verschiebung der Zellen, angeführt von medialen PSM-Zellen und gefolgt von lateral gelegenen Zellen. Diese anteriore Verschiebung geschieht gleichzeitig mit der posterioren Streckung des Embryos. Ein hypothetischer Schub durch neugebildete Zellen an der Schwanzspitze konnte ausgeschlossen werden. Mitotischen Zellen waren gleichmäßig in allen Geweben des Schwanzes verteilt und wiesen eine geringe Proliferationsrate auf. Die morphologischen Veränderungen und anterioren Verlagerungen des Schwanzmesoderms können daher hauptsächlich durch Zellmigration erklärt werden. Die Analysen konzentrierten sich daher auf die Zellmigration, insbesondere auf die zellulären Charakteristika, die sich während der Migration zeigen, wie z.B. Form, Orientierung, Volumen, Verteilung und Filopodienorganisation. So konnten neue Informationen darüber gewonnen werden, wie PSM-Zellen wandern und zur Somitenbildung beitragen. Die Nettobewegung der Schwanzverlängerung ist, unabhängig von nach anterior wandernden PSM-Zellen, nach posterior gerichtet. Das ist nur möglich, wenn einer lateralen Ausdehnung des PSM durch ungerichtet migrierenden Zellen entgegengewirkt wird. Über die Rolle einer laterale Begrenzung bei diesem Prozess gibt es bisher keine Untersuchungen. Beim Axolotl ist das PSM seitlich von einer zweischichtigen Epidermis und einer Fibronektin-reichen extrazellulären Matrix bedeckt. Nach Entfernung der Schwanzepidermis zeigten operierte Embryonen fehlende oder missgebildete Schwänze, insbesondere mit einer lateralen und dorsalen Krümmung und einer Verkürzung. Untersuchungen des Schwanzmesoderms zeigten ein erhöhtes PSM-Volumen und eine laterale Ausdehnung des Gewebes. Ein nahezu normaler Schwanz entwickelte sich, wenn die Embryonen nach Entfernung der Epidermis mit 1% Agarose, ergänzt mit Fibronektin, bedeckt wurden. Im Gegensatz dazu konnte eine einfache Abdeckung des PSM mit einer Nitrozellulosemembran, die Inkubation in der weicheren Methylzellulose oder in Agarose ohne Fibronektin die Schwanzbildung nicht normalisieren. Der seitliche Druck auf das PSM und eine Fibronektin-reiche extrazelluläre Matrix scheinen notwendig zu sein, um die Gewebearchitektur des PSM während der Schwanzbildung zu erhalten. Diese Studie zeigt das Verhalten einzelner PSM-Zellen während der Morphogenese der hinteren Neuralplatte bis zur Somitenbildung. Insgesamt konnte durch die spezifische Markierung von mesodermalen Zellen des Schwanzes deren Beitrag zur PSM-Morphologie aufgeklärt und ein detaillierteres Modell der Schwanzverlängerung vorgeschlagen werden: Die posteriore Ausdehnung des Neuralrohrs und des Notochords schiebt das posteriore Neuralplattengewebe nach hinten und quetscht die Zellen in eine verlängerte, mediolateral orientierte Form. Markierungsexperimente einzelner Zellgruppen zeigten, dass die ventralen, posterior gelegenen Zellen diesem Druck als erste entkommen, indem sie sich nach anterior verschieben. Ihnen folgen weiter anterior gelegene Zellen sowie dorsal gelegene Zellen. Diese Bewegungen erklären die anteriore Drehung. Dabei beginnen mesodermale Zellen ungerichtet zu wandern, verlängern sich und ändern ihre Orientierung von mediolateral nach anterior-posterior. Die ungerichtete Zellwanderung führt zu einer homogenen Zelldurchmischung, so dass zusammen mit dem PSM des Rumpfes ein einheitliches Gewebe gebildet wird. Die laterale Begrenzung durch die Epidermis kanalisiert die ungerichteten Migrationsbewegungen in anteriore Richtung. Auf diese Weise werden die Zellen in Richtung der Somitenbildungsstelle gelenkt, das PSM verengt sich, und der Embryo streckt sich nach hinten. Dieses Ausdehnungsmodell beinhaltet das individuelle Zellverhalten, das insgesamt die Morphologie des PSM prägt. Die analysierten dynamischen morphologischen Veränderungen des PSM können mit Schwanzentwicklungsprozessen und der posterioren Elongation der Achse in Verbindung gebracht werden.:1 Introduction 1.1 Embryonic tail formation . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1.1 Mechanism of tail formation . . . . . . . . . . . . . . . . . . . . . 1 1.1.2 Molecular determination of cell populations in the tail bud . . . . . 5 1.2 Axial elongation of the vertebrate body plan . . . . . . . . . . . . . . . . . 8 1.2.1 Anterior body elongation (elongation of the trunk) . . . . . . . . . 8 1.2.2 Posterior body elongation (tail elongation) . . . . . . . . . . . . . . 9 1.3 Studying tissue morphology during development . . . . . . . . . . . . 11 1.4 Aim of the project . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 12 2 Materials 2.1 Chemicals and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2 Antibodies and dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3 Techniqual equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3 Methods 3.1 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.1.1 Breeding of axolotls and embryo collection . . . . . . . . . . . . 19 3.1.2 Injections with the vital dye DiI . . . . . . . . . . . . . . . . . . . 19 3.1.3 Tissue transplantation techniques . . . . . . . . . . . . . . . . . . . 19 3.2 Immunohistochemical staining . . . . . . . . . . . . . . . . . . . . . . . . 20 3.2.1 Vibratome sections . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.2.2 Whole-mount staining . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.3 Optical tissue clearing protocols . . . . . . . . . . . . . . . . . . 21 3.3.1 Ethyl cinnamate based optical tissue clearing protocol . . . . . . . 21 3.3.2 SeeDB optical clearing protocol . . . . . . . . . . . . . . . . . . . . 22 3.4 Image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.4.1 3D image generation and processing . . . . . . . . . . .. . . . . . 22 3.4.2 Length measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.4.3 Manual segmentation . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4.4 Automatic segmentation . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5 Determination of cellular parameters . . . . . . . . . . . . . . .. . . . . 25 3.5.1 Cell shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5.2 Cell and tissue volume . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5.3 Cellular distribution . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.5.4 Closest neighbour analysis . . . . . . . . . . . . . . . . . . . . . . . 26 3.5.5 Cell orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.5.6 Length and orientation of filopodia . . . . . . . . . . . . . . . . . . 31 3.5.7 Distance of cells to a plane . . . . . . . . . . . . . . . . . . . . . . . 31 3.5.8 Mitotic rate and spindle orientation . . . . . . . . . . . . . . . . . 32 4 Results 4.1 The presomitic mesoderm is associated with axial elongation. . . . . . 33 4.1.1 Elongation of the body axis . . . . . . . . . . . . . . . . . . . . . . 33 4.1.2 Contribution of different tissues . . . . . . . . . . . . . . . . . . . . 34 4.1.3 Differential contribution of mesoderm and epidermis . . . . . . . . . 40 4.1.4 Dual potential of mesodermal progenitors . . . . . . . . . . . . . . . 42 4.1.5 Mesodermal tissue expansion . . . . . . . . . . . . . . . . . . . . . 46 4.2 Cellular behaviour influences mesodermal morphology . . . . . . . . . 50 4.2.1 Cell division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4.2.2 Positional changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.2.3 Cellular characteristics . . . . . . . . . . . . . . . . . . . . . . . . . 59 Cell shape changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Change of cell orientation . . . . . . . . . . . . . . . . . . . . . . . 61 Orientation of filopodia . . . . . . . . . . . . . . . . . . . . . . . . . 63 Cell distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 4.3 The epidermis fascilitates mesodermal tissue integrity . . . . . . .. . . . 67 4.3.1 Mesodermal tissue integrity . . . . . . . . . . . . . . . . . . . . . . 68 4.3.2 Malformed tails after epidermis removal . . . . . . . . . . . . . . . 70 4.3.3 Alteration in mesodermal tissue dimensions . . . . . . . . . . . . . 73 4.3.4 Alteration of cell density after epidermis removal . . . . . . . . . . 77 4.3.5 Rescue of tail formation . . . . . . . . . . . . . . . . . . . . . . . . 80 5 Discussion 5.1 Cell migration of the presomitic mesodermal cells . . . . . . . . .. . . . 85 5.1.1 Continuity of gastrulation movements . . . . . . . . . . . . . . . . . 85 5.1.2 Directed migration . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 5.1.3 Random cell migration . . . . . . . . . . . . . . . . . . . . . . . . . 88 5.1.4 Lateral mechanical constriction . . . . . . . . . . . . . . . . . . . . 90 5.2 Non-volumetric growth of the presomitic mesoderm . . . . . . . . . . . . . 91 5.3 Models of tail presomitic mesoderm formation . . . . . . . . . . . . . . . . 93

No description available.

<p>Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors and intracellular signaling proteins. Previous studies have implied a wide spectrum of Shb-mediated cellular responses, which motivated me to further investigate the role of Shb in differentiation and angiogenesis. Embryonic stem (ES) cells differentiate into endoderm and mesoderm from a bipotent mesendodermal cell population. Interregulatory signals between these germlayers are required for further specification. ES cells overexpressing Shb with an inactive SH2 domain (R522K-Shb) altered the expression of endodermal genes as a consequence of upregulated FGF expression. This response was enhanced by addition of activin A, suggesting a synergistic mechanism operative between FGF and activin A signaling in endoderm specification. To investigate a role for Shb in mesodermal specification, Shb knockout ES cells were established. These cells showed a reduced ability to form blood vessels after VEGF stimulation and delayed downregulation of genes associated with mesendoderm, indicating a reduced capacity for these cells to enter later stages.</p><p>To assess a role for Shb in tumor cell apoptosis, Shb expression was silenced in angiosarcoma endothelial cells. FAK-phosphorylation was reduced in Shb knockdown cells and this made them more susceptible to apoptotic stimuli both in vitro and in vivo.</p><p>Shb knockout microvasculature in mouse kidney, liver, and heart showed irregular endothelial linings with cytoplasmic projections toward the lumen, a feature that was also related to increased vascular permeability. VEGF treatment failed to stimulate vascular permeability in Shb knockout mice.</p><p>In order to elucidate whether these features relate to reduced angiogenesis, tumor growth was examined. Tumors grown in knockout mice showed reduced growth capacity and lower vessel density. In conclusion, Shb is a multifunctional adaptor protein that may be involved in several cellular responses both during embryonic development and adult life. </p>

Au cours des dernières décennies, les approches expérimentales nous ont permis d'obtenir des informations importantes en biologie du développement et nous ont conduit à la définition de réseaux complexes de régulation contrôlant les processus développementaux. Actuellement, notre compréhension de ces réseaux est entravée par leur complexité même. La modélisation mathématique est de plus en plus utilisée pour intégrer les voies de régulation et prévoir les effets de perturbations génétiques. Durant ma thèse, je me suis intéressée à la différentiation du mésoderme chez Drosophila melanogaster. Elle commence par la spécification du mésoderme en 4 différents tissus: le muscle viscéral, le coeur, le muscle somatique et le corps gras. La formation de ces tissus se traduit par une organisation segmentale répétitive le long du mésoderme. Mon premier but était de construire un modèle qui récapitule la spécification de ces quatre tissus entre les stades 8 et 10. Par la suite, je me suis concentrée sur le développement du coeur dans le but de proposer un modèle de régulation de la diversification des cellules cardiaques contractiles (cardioblastes) entre les stade 10 et 12. Afin de comprendre ces processus complémentaires, j'ai été amené à modéliser les voies de signalisation qui jouent un rôle important dans le développement du mésoderme et des cardioblastes. Je me suis appuyée sur des données génétiques et des analyses haut-débit publiées (HhIP-chip, ChIP-seq et transcriptome) pour déterminer et annoter des graphes de régulation complet pour chacun de ces réseaux ou voies. / During the past decades, experimental approaches have allowed us to gain important insights in developmental biology, and led to the delineation of complex regulatory networks controlling developmental processes. Currently, our understanding of these networks is hindered by their sheer complexity. Mathematical modelling is increasingly used to integrate regulatory pathways and predict the effects of genetic perturbations. My thesis focuses on the development of the specification of the mesoderm in Drosophila melanogaster. Its development results in the formation of different tissues segmentally iterated: the visceral muscle, the heart, the somatic muscle, and the fat body. My first goal was to build a network model recapitulating the specification of these 4 mesodermal tissues during stages 8 to 10. Then, focusing on heart development, my second aim was to build a network model recapitulating contractile cardiac cell (cardioblast) diversification during stages 10 to 12. To understand these complementary processes, I was further led to model the signalling pathways that play important roles in mesoderm and cardioblast development. I rely on a combination of published genetic data and high- throughput analyses (ChIP-chip, ChiP-seq, transcriptome) to delineate and annotate comprehensive regulatory graphs for each of these networks or pathways. Using a logical formalism and the GINsim software, I have further defined logical rules enabling the simulation of wild type and mutant behaviours for each of this networks or pathways. By and large, my model simulations recapitulate all relevant published data.

<p>The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation.</p><p>Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. </p><p>To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells.</p><p>Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers.</p><p>In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification. </p>

The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation. Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells. Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers. In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification.

Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors and intracellular signaling proteins. Previous studies have implied a wide spectrum of Shb-mediated cellular responses, which motivated me to further investigate the role of Shb in differentiation and angiogenesis. Embryonic stem (ES) cells differentiate into endoderm and mesoderm from a bipotent mesendodermal cell population. Interregulatory signals between these germlayers are required for further specification. ES cells overexpressing Shb with an inactive SH2 domain (R522K-Shb) altered the expression of endodermal genes as a consequence of upregulated FGF expression. This response was enhanced by addition of activin A, suggesting a synergistic mechanism operative between FGF and activin A signaling in endoderm specification. To investigate a role for Shb in mesodermal specification, Shb knockout ES cells were established. These cells showed a reduced ability to form blood vessels after VEGF stimulation and delayed downregulation of genes associated with mesendoderm, indicating a reduced capacity for these cells to enter later stages. To assess a role for Shb in tumor cell apoptosis, Shb expression was silenced in angiosarcoma endothelial cells. FAK-phosphorylation was reduced in Shb knockdown cells and this made them more susceptible to apoptotic stimuli both in vitro and in vivo. Shb knockout microvasculature in mouse kidney, liver, and heart showed irregular endothelial linings with cytoplasmic projections toward the lumen, a feature that was also related to increased vascular permeability. VEGF treatment failed to stimulate vascular permeability in Shb knockout mice. In order to elucidate whether these features relate to reduced angiogenesis, tumor growth was examined. Tumors grown in knockout mice showed reduced growth capacity and lower vessel density. In conclusion, Shb is a multifunctional adaptor protein that may be involved in several cellular responses both during embryonic development and adult life.

The mesoderm is the third germ layer, which is formed at gastrulation between the endoderm and the ectoderm in triploblastsc animals (Bilateria). The mesoderm differentiates into muscles, connective tissues and coelomic cavities. These structures have been key evolutionary innovations that prompted the enormous radiation of the bilaterians that at present make up for the 90% of animal species. As such, understanding the evolution of the mesoderm and its derivatives it is pivotal to understand the evolution of animals. In this thesis I have characterized the molecular patterning of the mesoderm and its derivatives (mainly muscles) in two different acoel species: Symsagittifera roscoffensis and Isodiametra pulchra. The acoels belong to the phylum Acoelomorpha (togheter with nemertodermatids and Xenoturbella). The phylogenetic placement of the Acoelomorpha is highly debated between a position basal to the bilaterians or nested inside the deuterostomes. The Acoelomorpha are morphologically simple animals and a trait sometimes considered a direct link to the cnidarians, the Bilateria sister group. With them they the acoelomorphs share a blind gut and a non centralized nervous system. Within the acoelomorphs, the acoels present the most derived body plan, however it is still rather simple if compared to other bilaterians. The nervous system for example is condensed anteriorly but not clear dorso ventral centralization exists as in most of the remaining bilaterians (the nerve cords are distributed circumferentially around the body). The mesoderm only develops from endodermal precursors, and this might be ancestral, since it is thought that the mesoderm evolved from the endoderm of a diploblastic, cnidarian-like ancestor. The muscles are the only mesodermal derivative in most basal acoelomorphs taxa, although in more advanced ones a parenchymal tissue, stem cells, and gonads also occupy the mesodermal space. The embryonic origins of the latter though, are at present still unknown. Thus acoelomorphs present most of traits considered to be eumetazoan ancestral traits (i.e. most of traits are also part of the cnidarians ground pattern), but still that the possibility that their body plan evolved in consequence of a secondary reduction must be considered as they could be more related to other deuterostomes than cnidarians. I have first investigated the molecular architecture of the muscles in the acoel Symsagittifera roscoffensis and found that although they have a smooth ultrastructural aspect they are molecularly more similar to the bilaterian striated muscles given that tey express key genes that control the contraction in the striated cells. This could be considered a first step into the evolution of the striated musculature without fully reaching it. Indeed, cnidarians have smooth epithelio muscular cells likely regulated by the same bilaterian smooth muscle proteins. However, the possibility of a secondary loss of the striation pattern cannot be discarded given that this already happened in some other bilaterians. Second, I have analyzed the expression of bilaterian mesodermal genes during embryogenesis and postembryonic development of Isodiametra pulchra and found that all but one (a FoxA ortholog) are expressed at the anterior pole, the site where the first myocytes start to differentiate. In juveniles and adults these genes are all expressed in muscles or at least a subset of them. Moreover the same genes are expressed in the gonads of I. pulchra and therefore it suggests that they could orginate in the endo-mesoderm of the worm. The cnidarians orthologues of these genes are expressed in the endoderm, which is moreover the site of the gametes differentation. The similarity between cnidarians endoderm and acoels mesoderm are astonishing, however before drawing conclusions we need a solid phylogenetic frame. / Los acelos son unos gusanos, principalmente marinos, de simetría bilateral y aplastados según el eje dorso ventral, que pertenecen al grupo de los acelomorfos (acelos +nemertodermatidos+xenoturbellidos), cuya posición filogenética es tema de debate entre los biólogos evolucionistas. Los acelomorfos carecen de cavidades corporales, su sistema digestivo es ciego y su sistema nervioso consiste de una concentración neuronal anterior y cuerda nerviosas no claramente desplazadas hacía el lado dorsal o ventral. La simplicidad morfológica de los acelos, entremedia entre la de cnidarios y bilaterales superiores, les hace buenos candidatos para el estudio de la transición de animales radiales-diploblastos a bilaterales-triploblastos. En esta tesis se presentan datos sobre el desarrollo e la especificación molecular del mesodermo, que ha sido una de las innovaciones claves para la radiación de los bilaterales. S. roscoffensis que como todos los acelos tiene exclusivamente musculatura de tipo liso, expresa un gen ortólogo a la troponina, un proteína clave para la regulación de los músculos estriados, y que no existe en cnidarios. La explicación más parsimoniosa es que las bases moleculares de evolución de músculos estriados se han implantado en los acelos, aunque estos no hayan alcanzado la condición completa (explicación favorecida si los acelomorfos son confirmados como grupo hermano de los demás bilaterales). Por otra parte se puede considerar esta condición como debida a una reducción secundaria (explicación favorecida en el caso que los acelos se confirmen ser deuteróstomos). Los ortólogos de genes endodermales de cnidarios y con clara expresión mesodermal en bilaterales se expresan en la músculatura del acelo l. pulchra. Estos datos concuerdan perfectamente con la evolución del mesodermo a partir del endodermo de animales diploblásticos. Aun así, es difícil proponer un modelo específico de evolución de miocitos hasta que la posición filogenética de los acelomorfos no esté resuelta.

One of the fundamental questions in developmental biology is how cells communicate during embryonic development to pattern the animal with defined axes and correctly placed organs. There are several key signal transduction pathways whose signaling has been found to be crucial during this period in the life history of many model organisms and whose functions have been well conserved between species. Two of those are the Notch and Hedgehog signal transduction pathways. Previous work established that the Notch pathway is important in the specification of mesoderm in the sea urchin embryo. Here it is established that the Hedgehog pathway is important for mesoderm patterning in the echinoderm embryo.In many animals, including the sea urchin, endomesoderm is specified as a bipotential tissue which is then subdivided through cell signaling to become endoderm and mesoderm. Notch signaling was found to be critical for that dichotomy; endomesoderm that received the Notch signal becomes mesoderm, the remaining endomesoderm becomes endoderm. Prior to this work, no functional roles for Hedgehog signaling in the sea urchin had been defined, though this pathway is known to operate in organisms throughout the animal kingdom. Here we find through analysis and comparison of the sea urchin genome with cnidarians, arthropods, urochordates, and vertebrates that key components and modifiers of the Notch and Hedgehog signaling pathways are well conserved among metazoans. Many animals contain the full suite of genes that constitute both pathways, and in deuterostomes the pathways operate in embryos to mediate similar fate decisions. The Notch pathway, for example, is engaged in endomesoderm gene regulatory networks and in neural functions. In the sea urchin RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm.The Hh signaling pathway is essential for patterning of many structures in vertebrates ranging from the nervous system, chordamesoderm, and limb to endodermal organs. In the sea urchin, a basal deuterostome, we show that Hedgehog (Hh) signaling participates in organizing the mesoderm. During gastrulation expression of the Hh ligand is localized to the endoderm while the co-receptors Patched (Ptc) and Smoothened (Smo) are expressed in the neighboring secondary mesoderm and in the ventrolaterally clustered primary mesenchyme cells where skeletogenesis initiates. Perturbations of Hh signaling cause embryos to develop with skeletal defects, as well as inappropriate secondary mesoderm patterning, although initial specification of secondary mesoderm occurs normally. Perturbations of Hedgehog pathway members altered normal numbers of pigment and blastocoelar cells, randomized left-right signaling in coelomic pouches, and resulted in disorganization of the circumesophageal muscle, causing an inability to perform peristaltic movements. Together our data support the requirement of Hh signaling in patterning each of the mesoderm subtypes in the sea urchin embryo.Activation of the Hedgehog pathway requires FoxA acting upstream of Hedgehog transcription, early in gastrulation. When FoxA is knocked-down there is a loss of transcription of Hedgehog and Hh expression is expanded in embryos expressing ectopic FoxA. In collaboration with another lab, we found that FoxA acts to repress mesodermal genes within the endoderm as part of the endomesoderm dichotomy. If FoxA expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, and less gut is specified. Conversely, when FoxA is ectopically expressed, endoderm is increased at the expense of mesoderm. More specifically we found through mosaic analysis that FoxA acts in a portion of the endomesoderm derived from one of two tiers of vegetal cells at the 60 cell stage called the veg2 cells. FoxA remains on in all endoderm and its territory of expression is superimposeable with the location of Hh expression.The data we present here together with previous studies suggest a model in which Notch signaling cues cells of the endomesoderm to become mesoderm, while cells of the nascent endoderm upregulate FoxA. FoxA ensures proper partitioning of endoderm from mesoderm by repressing mesoderm genes, as well as positively regulating transcription of Hedgehog in the endoderm. The Ptc and Smo transducing apparatus is separately expressed in mesoderm. Hh then signals to its receptors in the mesoderm to convey patterning information of tissues derived from that mesoderm. Thus, Hh, Ptc and Smo molecules diverge during specification then converge during signaling to play important roles in mesoderm development in the sea urchin. / Dissertation

In Drosophila melanogaster the visceral mesoderm (VM) develops during embryogenesis in a process where myoblasts become specified to generate two distinct cell types, the founder cells (FCs) and the fusion competent myoblasts (FCMs) that consequently fuses. The cell specification is dependent on cell signaling mediated by the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb), how this further sets up different identity programs that drive myoblasts to differentiate into FCs and FCMs is still not well understood. We have analysed whether the Midkine (MDK)/Pleiotrophin (PTN) homologues in Drosophila, Miple1 and Miple2 activate the Alk RTK in vivo. Earlier results from cell culture experiments suggested that vertebrate MDK/PTN is capable of activating ALK, findings that have become controversial with other studies showing contradictory results. We wanted to use Drosophila that have conserved homologues of both MDK/PTN and ALK, to address the question in vivo. We analysed the contribution of Miple in Alk dependent developmental processes such as visceral mesoderm (VM) specification during embryogenesis and in body size regulation of adult flies. Specification of VM as well as body size are not effected by loss of Miple proteins, and over expression of Miple proteins do not effect VM specification or body size. All together we conclude that there is no evidence that Miple1 or Miple2 can activate Alk in vivo. We found that loss of Miple protein effect the median lifespan of the fly which is reduced, interestingly the over expression of Miple proteins can promote an increased median life span in Drosophila. We have also analysed how Alk RTK signaling regulates the Gli-like transcription factor Lame duck (Lmd) in vivo on a post-translational level. It has already been reported that Lmd plays an essential role in specification of FCMs in the somatic mesoderm during embryogenesis. We detect Lmd protein exclusively in FCMs of VM in control embryos, but in Alk mutants Lmd protein is present in all cells of VM and opposite to this when Alk is activated in all cells in VM by over expression of Jeb this results in total loss of Lmd protein. This suggests that Alk signaling is regulating Lmd, and we additionally show that Lmd persist in FCMs in mutants where VM is specified but where myoblast fusion do not occur, supporting that Alk activity in FCs is regulating the downregulation of Lmd in FCMs upon fusion. Finally we have characterised the Rap1GEF C3G in vivo in Drosophila. In cell culture systems, the GTPase Rap1 has been identified to mediate Alk signaling and that this is regulated by the GEF C3G and interestingly the Drosophila C3G is expressed in the FCs of VM. We generated deletion mutants of C3G which exhibit semi-lethality and reduced life span, but no defects in visceral mesoderm development during embryogenesis. Instead we detected distinct phenotypes in somatic muscles of 3rd instar mutant larvae, with detachment and mistargeting of muscles, which effect localisation of integrins. We suggest that Drosophila C3G regulates Rap1 via inside out signaling of integrins which in turn effects cell adhesion in vivo in Drosophila larval muscles.