Rapid Wound Repair in the Ctenophore Mnemiopsis spp. by Matthew Brien Dodson A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements of the Degree of Masters Thesis Auburn, Alabama December 13, 2010 Keywords: invertebrate, wound repair, marine ctenophore, Mnemiopsis Copyright 2010 by Matthew Brien Dodson Approved by Anthony Moss, Chair, Associate Professor of Cell Biology Frank Bartol, Professor of Reproductive Biology Mark Liles, Assistant Professor of Microbiology ii Abstract The ability to quickly and efficiently close a wound is an integral part of any organism’s ability to survive. Marine organisms are exposed to numerous environmental pressures that demand the need for a rapid repair response. The delicate marine comb jelly Mnemiopsis leidyi faces damage from predation, as well as objects found in its natural surroundings, such as coarse sea grass and sediment. This damage presents the need to quickly and efficiently repair the wound to prevent further injury or infestation by degradative microbes that enter exposed mesoglea and gastrovascular canals. Mnemiopsis has evolved a rapid, scar-free wound repair mechanism that quickly seals the mesoglea away from the external environment. Here I describe the morphological changes that occur during repair, the development of a ctenophore wound cell culture system, and provide an initial look at gene expression during the repair response. ii Table of Contents Abstract........................................................................................................................................... ii List of Tables ...................................................................................................................................v List of Figures................................................................................................................................ vi Chapter 1: Wound Repair: A Review ..............................................................................................1 Importance of wound repair.................................................................................................1 Model systems and the conservation of wound repair.........................................................1 Repair at the cellular level ...................................................................................................3 Repair at the tissue level ......................................................................................................6 Regeneration and Wound Repair.......................................................................................20 Introduction to the phylum Ctenophora.............................................................................26 Chapter 2: Basic Light Microscopy Observations.........................................................................34 Introduction........................................................................................................................34 Objectives and Hypotheses................................................................................................35 Materials and Methods.......................................................................................................39 Results................................................................................................................................41 Discussion and Conclusions ..............................................................................................52 Chapter 3: Cell Culture Observations............................................................................................56 Introduction........................................................................................................................56 Objectives and Hypotheses................................................................................................57 iii Materials and Methods.......................................................................................................58 Results................................................................................................................................59 Discussion and Conclusions ..............................................................................................69 Chapter 4: Molecular Analyses of Wound Repair.........................................................................74 Introduction........................................................................................................................74 Objectives and Hypotheses................................................................................................81 Materials and Methods.......................................................................................................82 Results................................................................................................................................85 Discussion and Conclusions ..............................................................................................88 References......................................................................................................................................94 iv List of Tables Table 1: Quantification of cell morphologies present in vitro.......................................................67 Table 2: Cell sizes in vitro .............................................................................................................68 Table 3: Primer sequences .............................................................................................................84 Table 4: Functional significance of EST targets............................................................................85 Table 5: Degenerate primers..........................................................................................................86 v List of Figures Figure 1: Clot formation in Drosophila.........................................................................................10 Figure 2: Mnemiopsis leidyi...........................................................................................................28 Figure 3: Ctenophore oral-aboral axis ...........................................................................................29 Figure 4: Larval body plan of Mnemiopsis leidyi ..........................................................................29 Figure 5: Tissue organization.........................................................................................................30 Figure 6: Brightfield of an epithelial wound..................................................................................42 Figure 7: Cavity formation.............................................................................................................43 Figure 8: Hoechst staining .............................................................................................................44 Figure 9: Cells binding to muscle fibers........................................................................................45 Figure 10: Rearranging muscle fibers............................................................................................45 Figure 11: Actin rich wound edge .................................................................................................46 Figure 12: DAPI stain of wound edge ...........................................................................................47 Figure 13: Main cell morphologies at wound edge ......................................................................48 Figure 14: Cell network underlying the wound .............................................................................48 Figure 15: Change in epithelial cell polarity..................................................................................50 Figure 16: Cell dispersal ................................................................................................................51 Figure 17: Comparison of cutaneous repair rates ..........................................................................52 Figure 18: Progression of wound repair in Mnemiopsis ................................................................53 Figure 19: Cell culture in sea water ...............................................................................................61 vi Figure 20: Cell culture progression................................................................................................61 Figure 21: Cell morphologies observed in vitro ............................................................................62 Figure 22: Cell morphologies observed in vivo .............................................................................63 Figure 23: Bipolar cells in vitro.....................................................................................................64 Figure 24: Stellate cells in vitro.....................................................................................................65 Figure 25: Multipolar cells in vitro................................................................................................66 Figure 26: Pie chart of quantified in vitro cell types .....................................................................67 Figure 27: Histogram of sphere, spindle and bipolar cell sizes .....................................................68 Figure 28: Histogram of stellate and multipolar cell sizes ............................................................69