ABSTRACT
Epithelial remodeling determines the structure of many organs in the body through changes in cell shape, polarity and behavior and is a major area of study in developmental biology. Accurate and high-throughput methods are necessary to systematically analyze epithelial organization and dynamics at single-cell resolution. We developed SEGGA, an easy-to-use software for automated image segmentation, cell tracking and quantitative analysis of cell shape, polarity and behavior in epithelial tissues. SEGGA is free, open source, and provides a full suite of tools that allow users with no prior computational expertise to independently perform all steps of automated image segmentation, semi-automated user-guided error correction, and data analysis. Here we use SEGGA to analyze changes in cell shape, cell interactions and planar polarity during convergent extension in the Drosophila embryo. These studies demonstrate that planar polarity is rapidly established in a spatiotemporally regulated pattern that is dynamically remodeled in response to changes in cell orientation. These findings reveal an unexpected plasticity that maintains coordinated planar polarity in actively moving populations through the continual realignment of cell polarity with the tissue axes.
Subject(s)
Cell Polarity , Cytological Techniques/methods , Epithelial Cells/cytology , Software , Animals , Automation , Cell Shape , Cell Tracking , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Embryo, Nonmammalian/cytology , Epithelial Cells/metabolism , Genotype , Image Processing, Computer-AssistedABSTRACT
Cell rearrangements shape the Drosophila embryo via spatially regulated changes in cell shape and adhesion. We show that Bazooka/Par-3 (Baz) is required for the planar polarized distribution of myosin II and adherens junction proteins and polarized intercalary behavior is disrupted in baz mutants. The myosin II activator Rho-kinase is asymmetrically enriched at the anterior and posterior borders of intercalating cells in a pattern complementary to Baz. Loss of Rho-kinase results in expansion of the Baz domain, and activated Rho-kinase is sufficient to exclude Baz from the cortex. The planar polarized distribution of Baz requires its C-terminal domain. Rho-kinase can phosphorylate this domain and inhibit its interaction with phosphoinositide membrane lipids, suggesting a mechanism by which Rho-kinase could regulate Baz association with the cell cortex. These results demonstrate that Rho-kinase plays an instructive role in planar polarity by targeting Baz/Par-3 and myosin II to complementary cortical domains.
Subject(s)
Cell Polarity , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Embryo, Nonmammalian/physiology , Intracellular Signaling Peptides and Proteins/metabolism , rho-Associated Kinases/physiology , Animals , Animals, Genetically Modified , Blotting, Western , Body Patterning , Cell Nucleus/genetics , Cell Nucleus/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Female , Gene Expression Regulation, Developmental , Immunoenzyme Techniques , Intracellular Signaling Peptides and Proteins/genetics , Male , Myosin Type II/genetics , Myosin Type II/metabolism , Phosphorylation , Protein Kinase C/genetics , Protein Kinase C/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transgenes/physiologyABSTRACT
Elongation of the body axis is accompanied by the assembly of a polarized cytoarchitecture that provides the basis for directional cell behavior. We find that planar polarity in the Drosophila embryo is established through a sequential enrichment of actin-myosin cables and adherens junction proteins in complementary surface domains. F-actin accumulation at AP interfaces represents the first break in planar symmetry and occurs independently of proper junctional protein distribution at DV interfaces. Polarized cells engage in a novel program of locally coordinated behavior to generate multicellular rosette structures that form and resolve in a directional fashion. Actin-myosin structures align across multiple cells during rosette formation, and adherens junction proteins assemble in a stepwise fashion during rosette resolution. Patterning genes essential for axis elongation selectively affect the frequency and directionality of rosette formation. We propose that the generation of higher-order rosette structures links local cell interactions to global tissue reorganization during morphogenesis.
