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Cellular Interactions During Gastrulation in the Amphipod Crustacean, Parhyale Hawaiensis

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Cellular Interactions During Gastrulation in the Amphipod Crustacean, Parhyale Hawaiensis Cellular interactions during gastrulation in the amphipod crustacean, Parhyale hawaiensis by Ro Crystal Chaw A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Integrative Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Nipam H. Patel, chair Professor David Bilder Professor John Gerhart Professor Leslea Hlusko Fall 2011 Cellular interactions during gastrulation in the amphipod crustacean, Parhyale hawaiensis © 2011 by Ro Crystal Chaw Abstract Cellular interactions during gastrulation in the amphipod crustacean, Parhyale hawaiensis by Ro Crystal Chaw Doctor of Philosophy in Integrative Biology University of California, Berkeley Professor Nipam H. Patel, Chair How animals develop the complex structures and morphologies that result in diversification and speciation is a fundamental question in evolutionary biology that can be addressed through studies of embryogenesis. Gastrulation is an early morphogenetic event that establishes the embryonic tissue layers that will give rise to the various organs and characteristics of the adult. Cells and their interactions are the basis of this process. Outside of Drosophila melanogaster, little is known about the behavior of cells during gastrulation in arthropods. This dissertation focuses on cell behavior during gastrulation in the amphipod crustacean Parhyale hawaiensis. Using a variety of techniques, we make novel observations about cell shape change, the autonomy of cell behavior, and investigate possible molecular regulators of the entire process. I found cell shape change in the rosette that confirms previous hypotheses suggesting that gastrulation in Parhyale proceeds through ingression or invagination. I also found that the rosette and epithelial sheet act autonomously, and that the descendants of different lineages within the rosette are able to internalize independently. Using drug inhibition, I identified rho- kinase as important to epithelial integrity during gastrulation, and discovered a phenotype that may be associated with abnormal cell division. Finally, I investigated several candidate genes in an effort to discover a molecular marker for early mesoderm and ectoderm fate. This study provides valuable information about the process of cell internalization, cell autonomy during gastrulation, and suggests potential highly conserved mechanisms for cell behavior during Parhyale gastrulation. 1 This thesis is lovingly dedicated to my Dad, Shaw Yung Chaw (1947-2003). A constant companion on fishing expeditions, in cooking elaborate meals, and on long walks through neighborhoods and on mountains, my father was my partner in crime and my biggest cheerleader. I miss him every day. This dissertation is as much his as it is mine. i Table of Contents Abstract .......................................................................................................................... 1! Table of Contents...........................................................................................................ii! Table of Figures ............................................................................................................. v! Acknowledgements....................................................................................................... vi! Chapter I: Introduction ............................................................................................... 1! Cell Behavior and Gastrulation in model systems ..............................................................3! Bottle cells during Xenopus laevis gastrulation..................................................................3! Ingression during Sea urchin gastrulation (Strongylocentrotus spp., Lytechinus spp., Heliocidaris spp.) .................................................................................................................5! Ingression of the endoderm in Caenorhabditis elegans .......................................................5! Invagination of the mesoderm in Drosophila melanogaster ................................................6! Gastrulation in crustaceans and Parhyale ...........................................................................8! Chapter II: Cell shape change in the rosette during internalization...................... 16! Summary...............................................................................................................................16! Introduction ..........................................................................................................................16! Materials and Methods ........................................................................................................17! Husbandry, dissection, and fixation...................................................................................17! Staging table and cell tracking in time-lapse video ...........................................................17! Thick sectioning.................................................................................................................18! Antibody and phalloidin staining of whole embryos and sections ....................................18! Microinjection....................................................................................................................18! Results ...................................................................................................................................18! Timing of and movements during rosette formation and internalization ..........................18! Bottle cells in the rosette....................................................................................................19! The germline migrates inward after Mav ..........................................................................20! Discussion..............................................................................................................................20! Chapter III: Cellular interactions during rosette internalization. ......................... 32! Summary...............................................................................................................................32! Introduction ..........................................................................................................................32! Materials and Methods ........................................................................................................34! Photoablation .....................................................................................................................34! Manual ablation .................................................................................................................34! Cell tracing and analysis ....................................................................................................35! Results ...................................................................................................................................35! Mav and g can internalize independently ..........................................................................36! Rosette and epithelial sheet migration are autonomous: Rosette ablation.........................37! Rosette and epithelial sheet migration are autonomous: Ablation of the leading edge of epithelial cells ....................................................................................................................38! Discussion..............................................................................................................................39! Parhyale is able to gastrulate without the rosette or epithelial sheet.................................39! Yolk condensation vs. a leading edge signaling center .....................................................39! Chapter IV: Pharmacological inhibition of gastrulation........................................ 57! Summary...............................................................................................................................57! ii Introduction .............................................................................................................................. 57! Materials and Methods ........................................................................................................58! Microinjection....................................................................................................................58! Drug treatment ...................................................................................................................58! Antibody and phalloidin staining.......................................................................................58! Results ...................................................................................................................................58! Inhibition of microtubules with nocodazole, but not taxol, arrests gastrulation................58! Cytochalasin D inhibits gastrulation in Parhyale ..............................................................60! Inhibition with Olomoucine does not prevent gastrulation ...............................................61! Pharmocological inhibition of rho-kinase arrests gastrulation ..........................................61! Discussion..............................................................................................................................62! Inhibition of actin and rho-kinase arrests gastrulation.......................................................62! A
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