The interplay between biochemical signals and mechanical processes during animal development is key for the formation of tissues and organs with distinct shapes and functions. An important step during the formation of many tissues is the formation of compartment boundaries which separate cells of different fates and functions. Compartment boundaries are lineage restrictions that are characterized by a straight morphology. Biochemical signaling across compartment boundaries induce the expression of morphogens in the cells along the boundaries. These morphogens then act at long-range to direct growth and patterning of the whole tissue. Compartment boundaries stabilize the position of morphogens and thereby contribute to proper tissue development. The straight morphology of compartment boundaries is challenged by cell rearrangements caused by cell division and tissue reshaping. Physical mechanisms are therefore required to maintain the straight morphology of compartment boundaries. The anteroposterior (A/P) compartment boundary in the developing Drosophila melanogaster wing is established by biochemical signals. Furthermore, mechanical processes are required to maintain the straight shape of the A/P boundary. Recent studies show that mechanical tension mediated by actomyosin motor proteins is increased along the A/P boundary. However, it was not understood how biochemical signals interact with mechanical processes to maintain the A/P boundary. Here I provide the first evidence that Hedgehog signaling regulates mechanical tension along the A/P boundary. I was able to show that differences in Hedgehog (Hh) signal transduction activity between the anterior and posterior compartments are necessary and sufficient to maintain the straight shape of the A/P boundary, which is crucial for patterning and growth of the adult wing. Moreover, differences in Hh signal transduction activity are necessary and sufficient for the increase in mechanical tension along the A/P boundary. In addition, differences in Hh signal transduction activity are sufficient to generate smooth borders and to increase mechanical tension along ectopic interfaces. Furthermore, the differential expression of the transmembrane protein Capricious is sufficient to increase mechanical tension along ectopic interfaces. It was previously suggested that mechanical tension is generated by an actomyosin-cable through which the increase in mechanical tension is transmitted between the junctions along the A/P boundary. Here I show that mechanical tension is generated locally at each cell bond and not transmitted between junctions by an actomyosin cable. My results provide new insights for our understanding of the interplay between biochemical signals and mechanical processes during animal development.

Epithelial folding is an important morphogenetic process that is essential in transforming simple sheets of cells into complex three-dimensional tissues and organs during animal development (Davidson, 2012). Epithelial folding has been shown to rely on constriction forces generated by the apical actomyosin network (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). However, the contributions of mechanical forces acting along lateral and basal cell surfaces to epithelial folding remain poorly understood. Here we combine live imaging with force measurements of epithelial mechanics to analyze the formation of two epithelial folds in the Drosophila larval wing imaginal disc. We show that these two neighboring folds form via two distinct mechanisms. These two folds are driven either by decrease of basal tension or increase of lateral tension, none of them depends on apical constriction. In the first fold, a local decrease in extracellular matrix (ECM) density in prefold cells results in a reduction of mechanical tension on the basal cell surface, leading to basal expansion and fold formation. Consistent with that, a local reduction of ECM by overexpression of Matrix metalloproteinase II is sufficient to induce ectopic folding. In the second fold a different mechanism is at place. Here basal tension is not different with neighboring cells, but pulsed dynamic F-actin accumulations along the lateral interface of prefold cells lead to increased lateral tension, which drives cell shortening along the apical-basal axis and fold formation. In this thesis I described two distinct mechanisms driving epithelial folding, both basal decrease and lateral increase in tension can generate similar morphological changes and promote epithelial folding in the Drosophila wing discs. / Die Faltung von Epithelien ist ein wichtiger morphogenetischer Prozess, der die Entstehung komplexer, dreidimensionaler Gewebe und Organe aus einfachen Zellschichten ermöglicht (Davidson, 2012). Es ist bekannt, dass Kräfte erzeugt durch das apikale Aktomyosin-Netzwerk wichtig sind für die erfolgreiche Faltung von Epithelien (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). Die Rolle von mechanischen Kräften, die entlang der lateralen und basalen Seite wirken, ist jedoch kaum verstanden. Wir verbinden Lebendmikroskopie mit der Messung von mechanischen Eigenschaften, um die Entstehung von 2 Epithelfalten in den Imaginalscheiben von Drosophila zu verstehen. Wir können dadurch zeigen, dass die beiden Falten durch unterschiedliche Mechanismen entstehen. Sie entstehen entweder durch eine Verringerung der Spannung auf der basalen Seite oder durch eine Erhöhung der Spannung auf der lateralen Seite, aber keine von beiden entsteht durch zusammenziehende Kräfte auf der apikalen Seite. Die erste Falte entsteht durch eine lokale Verringerung der extrazellulären Matrix in den Vorläuferzellen, was zu einer Reduktion der Spannung auf der basalen Seite und zur Ausbildung der Falte führt. Die zweite Falte wird durch einen anderen Mechanismus ausgebildet. Hier ist nicht die Spannung auf der basalen Seite reduziert sondern dynamische Anreicherungen von F-Aktin auf der lateralen Seite resultieren in einer erhöhten lateralen Spannung, die zu einer Verkürzung der Zellen und damit zur Ausbildung einer Falte führt. In meiner Arbeit zeige ich 2 neue Mechanismen zur Entstehung von Epithelfalten auf, durch Absenken der Spannung auf der basalen oder Erhöhen auf der lateralen Seite.

Während der Gewebeentwicklung beeinflusst die Anpassung der mechanischen Spannung bei Zell-zu-Zell-Kontakten das Gewebewachstum, die Musterbildung und die Morphogenese. Die Erzeugung und Kontrolle der mechanischen Spannung hängt von Komponenten des Zytoske- letts wie dem Aktomyosin und den Mikrotubuli-Netzwerken ab. Die Bildung von Komparti- mentgrenzen ist ein wichtiger Entwicklungsprozess, der auf der Anpassung mechanischer Spannungen beruht. Kompartimentgrenzen sind Abstammungsbeschränkungen, die Zellen mit unterschiedlichen Funktionen und Identitäten innerhalb von Geweben trennen. Zellverbindun- gen entlang der Kompartimentgrenzen sind häufig durch eine Anreicherung von filamentösen (F-) Aktin sowie nicht-muskulären Myosin II (Myosin II) Motorprotein und erhöhter mechani- scher Spannung gekennzeichnet. Die Mechanismen, durch die F-Aktin und Myosin II an die- sen Verbindungsstellen angereichert werden, sind jedoch kaum verstanden. Hier zeigen wir, dass an der sich bildenden anteroposterioren Kompartimentgrenze der Puppenepidermis von Drosophila melanogaster F-Aktin und Myosin II vorübergehend angereichert werden. Die An- reicherung von F-Aktin scheint nicht von mechanischer Spannung abzuhängen. Die Fluores- zenzerholung nach Photobleichversuchen (Fluorescence recovery after photobleaching, FRAP) weist eher darauf hin, dass Myosin II vorzugsweise an Zellübergängen entlang der Komparti- mentgrenze stabilisiert wird. Darüber hinaus zeigen wir unter Verwendung einer photokonver- tierbaren Form von Myosin II, dass Myosin II vorzugsweise aus einem zytosolischen Pool an Zellverbindungen entlang der Kompartimentgrenze rekrutiert wird. Um die Rolle des Mikro- tubuli-Netzwerks bei der Bildung von Kompartimentgrenzen zu testen, haben wir außerdem dessen Organisation in der Puppenepidermis charakterisiert. Wir zeigen, dass sich Mikrotubuli und das Mikrotubuli-Minus-Ende-bindende Protein Patronin in einem Streifen anteriorer Zellen entlang der Kompartimentgrenze ansammeln. Interessanterweise haben die Zellen in diesem Streifen, im Vergleich zu anderen Zellen in der Epidermis, eine unterschiedliche Form. Zusammengefasst enthüllen unsere Daten Unterschiede in der Organisation der Mikrotubuli, die mit Kompartimentgrenzen verbunden sind, und zeigen, dass die Anreicherung von Myosin II entlang der Kompartimentgrenze der Puppen-Abdominalepidermis sowohl eine bevorzugte Stabilisierung als auch eine Rekrutierung beinhaltet.

La fasciculation des axones joue un rôle essentiel dans le développement des réseaux neuronaux. Cependant, la dynamique de la fasciculation axonale, ainsi que les mécanismes biophysiques à l’œuvre dans ce processus, demeurent encore très mal compris. En vue d'étudier les mécanismes de fasciculation d'axones ex vivo, nous avons développé un système modèle simple, constitué par des explants d'épithélium olfactif de souris embryonnaires en culture, à partir desquels poussent les axones des neurones sensoriels olfactifs. Grâce à une étude en vidéomicroscopie, nous avons observé que ces axones interagissent de façon dynamique par leur fibre, à la manière de fermetures éclair pouvant se fermer ("zippering") ou s'ouvrir ("unzippering"), ce qui conduit respectivement à la fasciculation ou à la défasciculation des axones. Mettant à profit cette nouvelle préparation expérimentale pour l'étude des interactions dynamiques entre axones, nous avons développé une analyse biophysique détaillée des processus de zippering/unzippering.Nous mettons en évidence dans notre travail l'existence d'un mécanisme biophysique cohérent de contrôle des interactions locales entre fibres axonales. Ce mécanisme local est à mettre en relation avec les changements de la structure globale du réseau axonal (degré de fasciculation) qui s'opèrent sur une échelle temporelle plus longue. Enfin, nous discutons la signification fonctionnelle de nos observations et analyses, et proposons un nouveau rôles de la tension mécanique dans le développement du système nerveux : la régulation de la fasciculation des axones et, en conséquence, de la formation des cartes topologiques au sein des réseaux neuronaux. / While axon fasciculation plays a key role in the development of neural networks, very little is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behaviour that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments.We show that there is a consistent mechanism which governs local interactions between axon shafts, supported by broad experimental evidence. This mechanism can be reconciled with changes in global structure of axonal network developing on slower time scale, analogically to well-studied relation between local relaxations, and topological changes and coarsening in two-dimensional liquid foams. We assess our observations and analysis in light of possible in vivo functional significance and propose a new role of mechanical tension in neural development: the regulation of axon fasciculation and consequently formation of neuronal topographic maps.

The interplay between biochemical signals and mechanical processes during animal development is key for the formation of tissues and organs with distinct shapes and functions. An important step during the formation of many tissues is the formation of compartment boundaries which separate cells of different fates and functions. Compartment boundaries are lineage restrictions that are characterized by a straight morphology. Biochemical signaling across compartment boundaries induce the expression of morphogens in the cells along the boundaries. These morphogens then act at long-range to direct growth and patterning of the whole tissue. Compartment boundaries stabilize the position of morphogens and thereby contribute to proper tissue development. The straight morphology of compartment boundaries is challenged by cell rearrangements caused by cell division and tissue reshaping. Physical mechanisms are therefore required to maintain the straight morphology of compartment boundaries. The anteroposterior (A/P) compartment boundary in the developing Drosophila melanogaster wing is established by biochemical signals. Furthermore, mechanical processes are required to maintain the straight shape of the A/P boundary. Recent studies show that mechanical tension mediated by actomyosin motor proteins is increased along the A/P boundary. However, it was not understood how biochemical signals interact with mechanical processes to maintain the A/P boundary. Here I provide the first evidence that Hedgehog signaling regulates mechanical tension along the A/P boundary. I was able to show that differences in Hedgehog (Hh) signal transduction activity between the anterior and posterior compartments are necessary and sufficient to maintain the straight shape of the A/P boundary, which is crucial for patterning and growth of the adult wing. Moreover, differences in Hh signal transduction activity are necessary and sufficient for the increase in mechanical tension along the A/P boundary. In addition, differences in Hh signal transduction activity are sufficient to generate smooth borders and to increase mechanical tension along ectopic interfaces. Furthermore, the differential expression of the transmembrane protein Capricious is sufficient to increase mechanical tension along ectopic interfaces. It was previously suggested that mechanical tension is generated by an actomyosin-cable through which the increase in mechanical tension is transmitted between the junctions along the A/P boundary. Here I show that mechanical tension is generated locally at each cell bond and not transmitted between junctions by an actomyosin cable. My results provide new insights for our understanding of the interplay between biochemical signals and mechanical processes during animal development.

Epithelial folding is an important morphogenetic process that is essential in transforming simple sheets of cells into complex three-dimensional tissues and organs during animal development (Davidson, 2012). Epithelial folding has been shown to rely on constriction forces generated by the apical actomyosin network (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). However, the contributions of mechanical forces acting along lateral and basal cell surfaces to epithelial folding remain poorly understood. Here we combine live imaging with force measurements of epithelial mechanics to analyze the formation of two epithelial folds in the Drosophila larval wing imaginal disc. We show that these two neighboring folds form via two distinct mechanisms. These two folds are driven either by decrease of basal tension or increase of lateral tension, none of them depends on apical constriction. In the first fold, a local decrease in extracellular matrix (ECM) density in prefold cells results in a reduction of mechanical tension on the basal cell surface, leading to basal expansion and fold formation. Consistent with that, a local reduction of ECM by overexpression of Matrix metalloproteinase II is sufficient to induce ectopic folding. In the second fold a different mechanism is at place. Here basal tension is not different with neighboring cells, but pulsed dynamic F-actin accumulations along the lateral interface of prefold cells lead to increased lateral tension, which drives cell shortening along the apical-basal axis and fold formation. In this thesis I described two distinct mechanisms driving epithelial folding, both basal decrease and lateral increase in tension can generate similar morphological changes and promote epithelial folding in the Drosophila wing discs. / Die Faltung von Epithelien ist ein wichtiger morphogenetischer Prozess, der die Entstehung komplexer, dreidimensionaler Gewebe und Organe aus einfachen Zellschichten ermöglicht (Davidson, 2012). Es ist bekannt, dass Kräfte erzeugt durch das apikale Aktomyosin-Netzwerk wichtig sind für die erfolgreiche Faltung von Epithelien (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). Die Rolle von mechanischen Kräften, die entlang der lateralen und basalen Seite wirken, ist jedoch kaum verstanden. Wir verbinden Lebendmikroskopie mit der Messung von mechanischen Eigenschaften, um die Entstehung von 2 Epithelfalten in den Imaginalscheiben von Drosophila zu verstehen. Wir können dadurch zeigen, dass die beiden Falten durch unterschiedliche Mechanismen entstehen. Sie entstehen entweder durch eine Verringerung der Spannung auf der basalen Seite oder durch eine Erhöhung der Spannung auf der lateralen Seite, aber keine von beiden entsteht durch zusammenziehende Kräfte auf der apikalen Seite. Die erste Falte entsteht durch eine lokale Verringerung der extrazellulären Matrix in den Vorläuferzellen, was zu einer Reduktion der Spannung auf der basalen Seite und zur Ausbildung der Falte führt. Die zweite Falte wird durch einen anderen Mechanismus ausgebildet. Hier ist nicht die Spannung auf der basalen Seite reduziert sondern dynamische Anreicherungen von F-Aktin auf der lateralen Seite resultieren in einer erhöhten lateralen Spannung, die zu einer Verkürzung der Zellen und damit zur Ausbildung einer Falte führt. In meiner Arbeit zeige ich 2 neue Mechanismen zur Entstehung von Epithelfalten auf, durch Absenken der Spannung auf der basalen oder Erhöhen auf der lateralen Seite.

Mechanical tension is an important regulator of cell proliferation, differentiation, migration and cell death. It is involved in the control of tissue architecture and wound repair and its improper sensing can contribute to cancer. The Hippo tumor suppressor pathway was recently shown to be involved in regulating cell proliferation in response to mechanical tension. The core of the pathway consists of the kinases MST1/2 and LATS1/2, which regulate the target of the pathway, the transcription co-activator YAP/ TAZ (hereafter referred to as YAP). When the Hippo pathway is inactive, YAP remains in the nucleus and promotes cell proliferation and stem cell maintenance. When the Hippo signaling pathway is turned on, MST1/2 phosphorylate and activates LATS1/2. LATS1/2 phosphorylates and inactivates YAP in the cytoplasm which is sequestered and degraded, stopping cell proliferation and promoting differentiation of stem cells. Mechanical forces are transmitted across cells and tissues through the cell-cell junctions and the actin cytoskeleton. However, the factors that connect cell-cell junctions to the Hippo signaling pathway were not clearly known. We identified a LIM domain protein called TRIP6 that functions at the adherens junctions to regulate the Hippo signaling pathway in a tension-dependent manner. TRIP6 responds to mechanical tension at adherens junctions and regulates LATS1/2 activity. Under high mechanical tension, TRIP6 sequesters and inhibits LATS1/2 at adherens junctions to promote YAP activity. Conditions that reduce tension at adherens junctions by inhibition of actin stress fibers or disruption of cell-cell junctions reduce TRIP6-LATS1/2 binding, which activates LATS1/2 to inhibit YAP. Vinculin has been shown to act as part of a mechanosensory complex at adherens junctions. We show that vinculin promotes TRIP6 inhibition of LATS1/2 in response to mechanical tension. Furthermore, we show that TRIP6 competitively inhibits MOB1 (a known LATS1/2 activator) from binding and activating LATS1/2. Together these findings reveal TRIP6 responds to mechanical signals at adherens junctions to regulate the Hippo signaling pathway in mammalian cells.

in English While axon fasciculation plays a key role in the development of neural networks, very lit- tle is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behaviour that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and me- chanical tension in the axons. This is upheld on quantitative level by conforming change of network global structure in response to various pharmacological treatments, without active involvement of growth cones. The calibrated manipulations of interacting shafts provide qualitative support for the hypothesis, and also allow us to quantify the mechan- ical tension of axons in our system. Furthermore, we introduce a biophysical model of the zippering dynamics, which efficiently serves the purpose of estimating the magnitude of remaining involved...