During development, cells consume energy, divide, rearrange, and die. Bulk properties such as viscosity and elasticity emerge from cell-scale mechanics and dynamics. Order appears, for example in patterns of hair outgrowth, or in the predominately hexagonal pattern of cell boundaries in the wing of a fruit fly. In the past fifty years, much progress has been made in understanding tissues as living materials. However, the physical mechanisms underlying tissue-scale behaviour are not completely understood. Here we apply theories from statistical physics and fluid dynamics to understand mechanics and order in two-dimensional tissues. We restrict our attention to the mechanics and dynamics of cell boundaries and vertices, and to planar polarity, a type of long-ranged order visible in anisotropic patterns of proteins and hair outgrowth. Our principle tool for understanding mechanics and dynamics is a vertex model where cell shapes are represented using polygons. We analytically derive the ground-state diagram of this vertex model, finding it to be dominated by the geometric requirement that cells be polygons, and the topological requirement that those polygons tile the plane. We present a simplified algorithm for cell division and growth, and furthermore derive a dynamic equation for the vertex model, which we use to demonstrate the emergence of quasistatic behaviour in the limit of slow growth. All our results relating to the vertex model are consistent with and build off past calculations and experiments. To investigate the emergence of planar polarity, we develop quantification methods for cell flow and planar polarity based on confocal microscope images of developing fly wings. We analyze cell flow using a velocity gradient tensor, which is uniquely decomposed into terms corresponding to local compression, shear, and rotations. We argue that a pattern in an inhomogeneously flowing tissue will necessarily be reorganized, motivating a hydrodynamic theory of polarity reorientation. Using such a coarse-grained theory of polarity reorientation, we show that the quantified patterns of shear and rotation in the wing are consistent with the observed polarity reorganization, and conclude that cell flow reorients planar polarity in the wing of the fruit fly. Finally, we present a cell-scale model of planar polarity based on the vertex model, unifying the themes of this thesis.

Planar cell polarity (PCP) refers to the coordinated orientation, movement, or structure of cells within the plane of a tissue. Zebrafish PCP mutants such as the vangl2 mutant exhibit defects in convergent extension, neural tube morphogenesis, and ciliary positioning. Tmem14a is a putative tetraspanin protein that was identified as an potential interactor of Vangl2 in a membrane yeast-two hybrid screen. GFP-tagged versions of Tmem14a are localized to the trans-Golgi network in zebrafish neuroepithelial cells. Knockdown of Tmem14a activity results in convergent extension defects, an ectopic accumulation of cells in the neural tube, and disorganized cilia. The localization of GFP-tagged Tmem14a to the trans-Golgi network suggested that Tmem14a plays a role in the trafficking of core PCP components to the cell membrane. Indeed, the membrane localization of GFP-Vangl2 was disrupted in Tmem14a morphants. Thus, Tmem14a is an interactor of Vangl2 and a novel regulator of vertebrate planar cell polarity signaling.

Planar cell polarity (PCP) refers to the coordinated orientation, movement, or structure of cells within the plane of a tissue. Zebrafish PCP mutants such as the vangl2 mutant exhibit defects in convergent extension, neural tube morphogenesis, and ciliary positioning. Tmem14a is a putative tetraspanin protein that was identified as an potential interactor of Vangl2 in a membrane yeast-two hybrid screen. GFP-tagged versions of Tmem14a are localized to the trans-Golgi network in zebrafish neuroepithelial cells. Knockdown of Tmem14a activity results in convergent extension defects, an ectopic accumulation of cells in the neural tube, and disorganized cilia. The localization of GFP-tagged Tmem14a to the trans-Golgi network suggested that Tmem14a plays a role in the trafficking of core PCP components to the cell membrane. Indeed, the membrane localization of GFP-Vangl2 was disrupted in Tmem14a morphants. Thus, Tmem14a is an interactor of Vangl2 and a novel regulator of vertebrate planar cell polarity signaling.

The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. / This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

We sought to investigate the role of the Planar Cell Polarity (PCP) pathway in neuronal positioning along the Anterior-Posterior (AP) axis of C. elegans, and chose the worm’s DD-type motor neurons as a model. The six DD neurons (DD1-DD6) are evenly spaced in the ventral nerve cord of wild type animals. Here we showed that mutations in core PCP genes caused DD neuron spacing and positioning defects. prkl-1 double mutant combinations with vang-1 and fmi-1 showed a suppression of the more severe prkl-1 single mutant defects, which was evidence of genetic interactions between these PCP components. We also conducted a candidate screen of Frizzled, Dishevelled, Wnt, and ROCK genes, and found that dsh-1/Dishevelled, mom-2/Wnt and let-502/ROCK also played roles in DD neuronal positioning. Both vang-1 and prkl-1 were found to function within the nervous system to guide DD neuronal positioning, and prkl-1 was further identified as playing a cell autonomous role. The origins of observed DD neuron anterior positioning defects were investigated during embryogenesis, in which 1.5 fold stage prkl-1(ok3182) embryos displayed delayed intercalation of the DD neurons. This represents a novel role for the PCP pathway in mediating DD neuronal intercalation.

During development, cells consume energy, divide, rearrange, and die. Bulk properties such as viscosity and elasticity emerge from cell-scale mechanics and dynamics. Order appears, for example in patterns of hair outgrowth, or in the predominately hexagonal pattern of cell boundaries in the wing of a fruit fly. In the past fifty years, much progress has been made in understanding tissues as living materials. However, the physical mechanisms underlying tissue-scale behaviour are not completely understood. Here we apply theories from statistical physics and fluid dynamics to understand mechanics and order in two-dimensional tissues. We restrict our attention to the mechanics and dynamics of cell boundaries and vertices, and to planar polarity, a type of long-ranged order visible in anisotropic patterns of proteins and hair outgrowth. Our principle tool for understanding mechanics and dynamics is a vertex model where cell shapes are represented using polygons. We analytically derive the ground-state diagram of this vertex model, finding it to be dominated by the geometric requirement that cells be polygons, and the topological requirement that those polygons tile the plane. We present a simplified algorithm for cell division and growth, and furthermore derive a dynamic equation for the vertex model, which we use to demonstrate the emergence of quasistatic behaviour in the limit of slow growth. All our results relating to the vertex model are consistent with and build off past calculations and experiments. To investigate the emergence of planar polarity, we develop quantification methods for cell flow and planar polarity based on confocal microscope images of developing fly wings. We analyze cell flow using a velocity gradient tensor, which is uniquely decomposed into terms corresponding to local compression, shear, and rotations. We argue that a pattern in an inhomogeneously flowing tissue will necessarily be reorganized, motivating a hydrodynamic theory of polarity reorientation. Using such a coarse-grained theory of polarity reorientation, we show that the quantified patterns of shear and rotation in the wing are consistent with the observed polarity reorganization, and conclude that cell flow reorients planar polarity in the wing of the fruit fly. Finally, we present a cell-scale model of planar polarity based on the vertex model, unifying the themes of this thesis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23571号 / 医博第4785号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 大森 孝一, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood. In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.