Animal morphogenesis is largely driven by concerted changes in the shape of individual cells. However, how cell shape changes are regulated and coordinated in developing animals is not well understood. Here we show that the two perpendicular signaling gradients of the morphogens Dpp, a TGF-β homologue, and Wingless, a Wnt family member, maintain tissue homoeostasis and control cell shape changes in the developing Drosophila wing. Clones of cells lacking Dpp or Wingless signaling invaginate apically, shorten apico-basally and subsequently extrude basally without disruption of the epithelium. During early larval development, the onset of Dpp and Wingless signaling correlates with the cuboidal-to-columnar cell shape transition of wing disc cells. Gradients in apical-basal length of columnar cells correlate during late larval development with the gradients of Dpp and Wingless signaling activities. Cells receiving high levels of Dpp and Wingless signaling are most elongated and apically constricted. Low levels of Dpp and Wingless signaling correlate with a shorter and apically wider cell morphology. Dpp and Wingless signaling is cell-autonomously required for maintaining the elongated columnar cell shape of late larval wing disc cells. Overactivation of these pathways results in precocious cell elongation during early larval development. These morphogenetic responses to Dpp and Wingless require the transcription factor complexes Mad and Tcf/β-catenin, respectively, indicating that they are mediated by changes in gene expression. The morphogenetic function of Wingless is in part mediated by one of its target genes, the transcription factor Vestigial. Wingless signaling promotes an enrichment of E-cadherin at the adherens junctions, and we show that E-cadherin is required to maintain apical-basal cell length. Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, we demonstrate that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. Our results identify cell-autonomous roles for Dpp and Wingless signaling in promoting and maintaining the elongated columnar shape of wing disc cells. Furthermore, they suggest that Dpp and Wingless signaling control cell shape by regulating the actin-MyosinII/E-cadherin network. / Morphogenese in Tieren wird in hohem Maße von konzertierten Zellformveränderungen einzelner Zellen bewirkt. Es ist jedoch noch nicht hinreichend verstanden, wie Zellformveränderungen in sich entwickelnden Tieren reguliert und koordiniert werden. Hier zeigen wir, dass die zwei zueinander senkrecht stehenden Signalgradienten der Morphogene Dpp, eines TGF-β Homologs, und Wingless, eines Mitglieds der Wnt Familie, im sich entwickelnden Drosophila-Flügel Gewebe-Homöostase aufrechterhalten und Zellformveränderungen kontrollieren. Klone von Zellen, denen Dpp oder Wingless Signalaktivität fehlt, invaginieren von ihrer apikalen Seite her, verkürzen sich in apiko-basaler Richtung und extruieren im Folgenden auf der basalen Seite des Epithels, ohne es zu zerstören. Während der frühen Larvalentwicklung korreliert das Anschalten der Dpp und Wingless Signale mit der Zellformveränderung der Flügelscheibenzellen von kuboidal zu kolumnar. Gradienten in der apiko-basalen Länge von kolumnaren Zellen korrelieren während der späten Larvalentwicklung mit den Gradienten der Dpp und Wingless Signalaktivitäten. Zellen, die hohe Werte an Dpp und Wingless Signalen empfangen, sind am meisten elongiert und apikal konstringiert. Niedrige Werte von Dpp und Wingless Signalen korrelieren mit kürzerer und apikal weiterer Zellmorphologie. Dpp und Wingless Signale werden zellautonom gebraucht für die Aufrechterhaltung der elongierten Zellform von späten larvalen Flügelscheibenzellen. Die Überaktivierung dieser Signalwege führt zu vorzeitiger Zellverlängerung während der frühen Larvalentwicklung. Diese morphogenetischen Antworten auf Dpp und Wingless benötigen die Transkriptionsfaktor-Komplexe Mad beziehungsweise Tcf/β-catenin, was darauf hindeutet, dass sie durch Änderungen in der Genexpression vermittelt werden. Die morphogenetische Funktion von Wingless wird teilweise durch eines seiner Zielgene, Vestigial, vermittelt. Wingless Signale fördern die Anreicherung von E-cadherin an den Adherensverbindungen. Wir zeigen hier, dass E-cadherin gebraucht wird, um apiko-basale Zelllänge aufrechtzuerhalten. Dpp Signale kontrollieren die subzelluläre Verteilung der Aktivitäten der kleinen GTPase Rho1 und der regulatorischen leichten Kette von nicht-muskulärem Myosin II (MRLC). Eine Änderung in der Rho1 oder MRLC Aktivität hat weitreichende Auswirkungen auf die apiko-basale Zelllänge. Schließlich zeigen wir noch, dass eine Verringerung der Rho1 oder MRLC Aktivitäten die Zellverkürzung von Dpp-Signal kompromittierten Zellen rettet. Unsere Resultate identifizieren zellautonome Rollen für Dpp und Wingless Signale in der Förderung und Aufrechterhaltung der elongierten kolumnaren Zellform von Flügelimaginalscheibenzellen. Darüber hinaus suggerieren sie, dass Dpp und Wingless Signale die Zellform durch die Regulierung des Aktin-MyosinII/E-cadherin-Netzwerks kontrollieren.

Animal morphogenesis is largely driven by concerted changes in the shape of individual cells. However, how cell shape changes are regulated and coordinated in developing animals is not well understood. Here we show that the two perpendicular signaling gradients of the morphogens Dpp, a TGF-β homologue, and Wingless, a Wnt family member, maintain tissue homoeostasis and control cell shape changes in the developing Drosophila wing. Clones of cells lacking Dpp or Wingless signaling invaginate apically, shorten apico-basally and subsequently extrude basally without disruption of the epithelium. During early larval development, the onset of Dpp and Wingless signaling correlates with the cuboidal-to-columnar cell shape transition of wing disc cells. Gradients in apical-basal length of columnar cells correlate during late larval development with the gradients of Dpp and Wingless signaling activities. Cells receiving high levels of Dpp and Wingless signaling are most elongated and apically constricted. Low levels of Dpp and Wingless signaling correlate with a shorter and apically wider cell morphology. Dpp and Wingless signaling is cell-autonomously required for maintaining the elongated columnar cell shape of late larval wing disc cells. Overactivation of these pathways results in precocious cell elongation during early larval development. These morphogenetic responses to Dpp and Wingless require the transcription factor complexes Mad and Tcf/β-catenin, respectively, indicating that they are mediated by changes in gene expression. The morphogenetic function of Wingless is in part mediated by one of its target genes, the transcription factor Vestigial. Wingless signaling promotes an enrichment of E-cadherin at the adherens junctions, and we show that E-cadherin is required to maintain apical-basal cell length. Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, we demonstrate that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. Our results identify cell-autonomous roles for Dpp and Wingless signaling in promoting and maintaining the elongated columnar shape of wing disc cells. Furthermore, they suggest that Dpp and Wingless signaling control cell shape by regulating the actin-MyosinII/E-cadherin network. / Morphogenese in Tieren wird in hohem Maße von konzertierten Zellformveränderungen einzelner Zellen bewirkt. Es ist jedoch noch nicht hinreichend verstanden, wie Zellformveränderungen in sich entwickelnden Tieren reguliert und koordiniert werden. Hier zeigen wir, dass die zwei zueinander senkrecht stehenden Signalgradienten der Morphogene Dpp, eines TGF-β Homologs, und Wingless, eines Mitglieds der Wnt Familie, im sich entwickelnden Drosophila-Flügel Gewebe-Homöostase aufrechterhalten und Zellformveränderungen kontrollieren. Klone von Zellen, denen Dpp oder Wingless Signalaktivität fehlt, invaginieren von ihrer apikalen Seite her, verkürzen sich in apiko-basaler Richtung und extruieren im Folgenden auf der basalen Seite des Epithels, ohne es zu zerstören. Während der frühen Larvalentwicklung korreliert das Anschalten der Dpp und Wingless Signale mit der Zellformveränderung der Flügelscheibenzellen von kuboidal zu kolumnar. Gradienten in der apiko-basalen Länge von kolumnaren Zellen korrelieren während der späten Larvalentwicklung mit den Gradienten der Dpp und Wingless Signalaktivitäten. Zellen, die hohe Werte an Dpp und Wingless Signalen empfangen, sind am meisten elongiert und apikal konstringiert. Niedrige Werte von Dpp und Wingless Signalen korrelieren mit kürzerer und apikal weiterer Zellmorphologie. Dpp und Wingless Signale werden zellautonom gebraucht für die Aufrechterhaltung der elongierten Zellform von späten larvalen Flügelscheibenzellen. Die Überaktivierung dieser Signalwege führt zu vorzeitiger Zellverlängerung während der frühen Larvalentwicklung. Diese morphogenetischen Antworten auf Dpp und Wingless benötigen die Transkriptionsfaktor-Komplexe Mad beziehungsweise Tcf/β-catenin, was darauf hindeutet, dass sie durch Änderungen in der Genexpression vermittelt werden. Die morphogenetische Funktion von Wingless wird teilweise durch eines seiner Zielgene, Vestigial, vermittelt. Wingless Signale fördern die Anreicherung von E-cadherin an den Adherensverbindungen. Wir zeigen hier, dass E-cadherin gebraucht wird, um apiko-basale Zelllänge aufrechtzuerhalten. Dpp Signale kontrollieren die subzelluläre Verteilung der Aktivitäten der kleinen GTPase Rho1 und der regulatorischen leichten Kette von nicht-muskulärem Myosin II (MRLC). Eine Änderung in der Rho1 oder MRLC Aktivität hat weitreichende Auswirkungen auf die apiko-basale Zelllänge. Schließlich zeigen wir noch, dass eine Verringerung der Rho1 oder MRLC Aktivitäten die Zellverkürzung von Dpp-Signal kompromittierten Zellen rettet. Unsere Resultate identifizieren zellautonome Rollen für Dpp und Wingless Signale in der Förderung und Aufrechterhaltung der elongierten kolumnaren Zellform von Flügelimaginalscheibenzellen. Darüber hinaus suggerieren sie, dass Dpp und Wingless Signale die Zellform durch die Regulierung des Aktin-MyosinII/E-cadherin-Netzwerks kontrollieren.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.