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During development of tissues, cells collectively organize to form complex patterns and morphologies. A general feature of many developing epithelia is their distinct organization into cellular compartments of different cell lineages. The interfaces between these compartments, called compartment boundaries, maintain straight and sharp morphologies. The interfaces play key roles in tissue development and pattern formation. An important model system to study the morphology of compartment boundaries during development is the wing disc of the fruit fly. Two compartment boundaries exist in the fly wing disc, the anteroposterior (AP) boundary and the dorsoventral (DV) boundary. A crucial question is how compartment boundaries are shaped and remain stable during growth. In this work, we discuss the dynamics and mechanisms of compartment boundaries in developing epithelia. We analyze the general features of interfacial phenomena in coarse- grained models of passive and active fluids. We introduce a continuum description of tissues with two cell types. This model allows us to study the propagation of interfaces due to the interplay of cell dynamics and tissue mechanics. We also use a vertex model to describe cellular compartments in growing epithelia. The vertex model accounts for cell mechanics and describes a 2D picture of tissues where the network of adherens junctions characterizes cell shapes. We use this model to study the general physical mechanisms by which compartment boundaries are shaped. We quantify the stresses in the cellular network and discuss how cell mechanics and growth influence the stress profile. With the help of the anisotropic stress profile near the interfaces we calculate the interfacial tension. We show that cell area pressure, cell proliferation rate, orientation of cell division, cell elongation created by external stress, and cell bond tension all have distinct effects on the morphology of interfaces during tissue growth. Furthermore, we investigate how much different mechanisms contribute to the effective interfacial tension. We study the mechanisms shaping the DV boundary in wing imaginal disc at different stages during the development. We analyze the images of wing discs to quantify the roughness of the DV boundary and average cell elongation in its vicinity. We quantify increased cell bond tension along the boundary and analyze the role of localized reduction in cell proliferation on the morphology of the DV boundary. We use experimentally determined values for cell bond tension, cell elongation and bias in orientation of cell division in simulations of tissue growth in order to reproduce the main features of the time-evolution of the DV boundary shape.

La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4α-phorbol 12,13-didecanoate (4α-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar. / Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4α- phorbol 12,13- idecanoate (4α-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.

During development of tissues, cells collectively organize to form complex patterns and morphologies. A general feature of many developing epithelia is their distinct organization into cellular compartments of different cell lineages. The interfaces between these compartments, called compartment boundaries, maintain straight and sharp morphologies. The interfaces play key roles in tissue development and pattern formation. An important model system to study the morphology of compartment boundaries during development is the wing disc of the fruit fly. Two compartment boundaries exist in the fly wing disc, the anteroposterior (AP) boundary and the dorsoventral (DV) boundary. A crucial question is how compartment boundaries are shaped and remain stable during growth. In this work, we discuss the dynamics and mechanisms of compartment boundaries in developing epithelia. We analyze the general features of interfacial phenomena in coarse- grained models of passive and active fluids. We introduce a continuum description of tissues with two cell types. This model allows us to study the propagation of interfaces due to the interplay of cell dynamics and tissue mechanics. We also use a vertex model to describe cellular compartments in growing epithelia. The vertex model accounts for cell mechanics and describes a 2D picture of tissues where the network of adherens junctions characterizes cell shapes. We use this model to study the general physical mechanisms by which compartment boundaries are shaped. We quantify the stresses in the cellular network and discuss how cell mechanics and growth influence the stress profile. With the help of the anisotropic stress profile near the interfaces we calculate the interfacial tension. We show that cell area pressure, cell proliferation rate, orientation of cell division, cell elongation created by external stress, and cell bond tension all have distinct effects on the morphology of interfaces during tissue growth. Furthermore, we investigate how much different mechanisms contribute to the effective interfacial tension. We study the mechanisms shaping the DV boundary in wing imaginal disc at different stages during the development. We analyze the images of wing discs to quantify the roughness of the DV boundary and average cell elongation in its vicinity. We quantify increased cell bond tension along the boundary and analyze the role of localized reduction in cell proliferation on the morphology of the DV boundary. We use experimentally determined values for cell bond tension, cell elongation and bias in orientation of cell division in simulations of tissue growth in order to reproduce the main features of the time-evolution of the DV boundary shape.

Indiana University-Purdue University Indianapolis (IUPUI) / While breast cancer is the second most commonly diagnosed cancer worldwide, its causes and natural history are not well defined. The female mammary organ is unique in that it does not reach full maturity until the lactation cycle following pregnancy. This cycle entails extensive growth and reorganization of the primitive epithelial ductal network. Following lactation, these same epithelial cells undergo an equally extensive program of apoptosis and involution. The mammary gland's sensitivity to pro-growth and pro-apoptotic signals may partly explain its proclivity to develop cancers. For epithelial cells to become transformed they must lose intracellular organization known as polarity as differentiated epithelial tissues are refractory to aberrant growth. One essential component of epithelial to mesenchymal transition is the intrinsic capacity of cells to repurpose polarity constituents to promote growth. Recently, a novel mechanism of organ size control has been shown to repurpose the apical junctional associated protein Yap into the nucleus where it functions as a transcriptional coactivator promoting growth and dedifferentiation. The focus of my work has been on a family of adaptor proteins termed Amots that have been shown to scaffold Yap and inhibit growth signaling. Specifically, I have shown that the 80KDa form of Amot, termed Amot80, acts as a dominant negative to the other Amot proteins to promote cell growth while reducing cell differentiation. Amot80 was found to promote the prolonged activation of MAPK signaling. Further, Amot80 expression was also found to enhance the transcriptional activity of Yap. This effect likely underlies the ability of Amot80 to drive disorganized overgrowth of MCF10A cells grown in Matrigel̈™. Overall, these data suggest a mechanism whereby the balance of Amot proteins controls the equilibrium between growth and differentiation within mammary epithelial tissues.