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Calcium Imaging of the Entire Muscle System of Hydra Reveals Extensive Cellular Multifunctionality

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Calcium Imaging of the Entire Muscle System of Hydra Reveals Extensive Cellular Multifunctionality Calcium imaging of the entire muscle system of Hydra reveals extensive cellular multifunctionality John Szymanski Submitted in partial fulfillment of the Requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences C O L U M B I A U N I V E R S I T Y 2018 © 2018 John Szymanski All rights reserved ABSTRACT Calcium imaging of the entire muscle system of Hydra reveals extensive cellular multifunctionality John Szymanski Hydra vulgaris is a cnidarian species with a life cycle containing only a polyp stage, and a simple body plan in which ectodermal and endodermal epitheliomuscular cells play many roles. One of their roles is to act as muscle—these two epithelia generate motion of the polyp by exerting contractile force through myonemes. The function of this musculature was studied on a system scale using whole-animal calcium imaging to measure functional activity in all epitheliomuscular cells simultaneously. This approach maps the diversity of functional activation patterns underlying the behavior of Hydra , and reveals that individual epitheliomuscular cells participate in multiple patterns using at least two different types of calcium signaling that propagate at vastly different speeds. These studies establish the functional basis for the epithelial muscle systems of Cnidaria, revealing new operational principles and deep evolutionary ties to mechanisms of contractile activity that exist elsewhere in Metazoa. CONTENTS LIST OF CHARTS, GRAPHS, ILLUSTRATIONS ..................................................................... iii ACKNOWLEDGEMENTS ........................................................................................................... iv INTRODUCTION .......................................................................................................................... 1 Anatomy of Hydra ....................................................................................................................... 2 Muscle diversity in Metazoa, Cnidaria, and Hydra ..................................................................... 4 Neuromuscular anatomy, dynamics, and behavior in Hydra ...................................................... 7 Behavior .................................................................................................................................. 7 Neuromuscular anatomy of Hydra .......................................................................................... 8 Electrophysiology ................................................................................................................... 9 Voltage-gated channels ......................................................................................................... 10 Neurotransmission ................................................................................................................ 12 The enteron: gastric cavity, hydrostatic skeleton, and osmoregulatory organ .......................... 14 Studies of muscle on the system scale ...................................................................................... 17 RESULTS ..................................................................................................................................... 19 Imaging the complete muscle activity of Hydra ....................................................................... 19 Diversity of patterns of epithelial calcium activity ................................................................... 23 Contraction bursts ................................................................................................................. 26 Column elongation ................................................................................................................ 28 Bending ................................................................................................................................. 29 Nodding................................................................................................................................. 30 Tentacle Contraction ............................................................................................................. 31 Slow wave ............................................................................................................................. 32 Mouth opening ...................................................................................................................... 32 Relationship of motion with calcium influx in contraction bursts ............................................ 34 Contraction burst initiation dynamics ....................................................................................... 37 Propagation velocity of slow waves .......................................................................................... 41 DISCUSSION ............................................................................................................................... 43 Extensive cellular multifunctionality in Hydra muscular system ............................................. 43 Mechanisms of activity propagation across Metazoa................................................................ 44 Dual epithelial activation in contraction bursts and osmoregulation ........................................ 47 CONCLUSION & FUTURE DIRECTIONS ............................................................................... 48 EXPERIMENTAL PROCEDURES ............................................................................................. 50 Hydra culture ............................................................................................................................. 50 Transgenics and grafting ........................................................................................................... 50 Imaging and image processing .................................................................................................. 51 i Imaging hardware ................................................................................................................. 51 Image processing .................................................................................................................. 51 Calculation of mean fluorescence traces ............................................................................... 51 Simultaneous suction electrode recording and calcium imaging .............................................. 52 REFERENCES ............................................................................................................................. 53 APPENDIX ................................................................................................................................... 62 ii LIST OF CHARTS, GRAPHS, ILLUSTRATIONS Figure 1: Anatomy of Hydra ......................................................................................................................................... 3 Figure 2: The Neuromuscular system of Hydra ............................................................................................................ 6 Figure 3: Gap junction connectivity between epitheliomuscular cells .......................................................................... 9 Figure 4: Distribution of expression of various HyNac Peptide-gated cation channels............................................... 13 Figure 5: Transgenics and imaging .............................................................................................................................. 22 Figure 6: Survery of patterns of epithelial activity ...................................................................................................... 25 Figure 7: Mean fluorescence traces during contraction bursts..................................................................................... 27 Figure 8: Mean fluorescence traces during active elongation ...................................................................................... 28 Figure 9: Mean fluorescence traces during bending .................................................................................................... 30 Figure 10: Mean fluorescence traces during nodding .................................................................................................. 30 Figure 11: Mean fluorescence traces during tentacle contraction ................................................................................ 31 Figure 12: Mean fluorescence traces during body column slow wave ........................................................................ 32 Figure 13: Relationship of motion with calcium influx ............................................................................................... 36 Figure 14: Contraction burst initiation dynamics ........................................................................................................ 38 Figure 15: Wavefront propagation speed of slow waves in ectoderm ......................................................................... 41 Figure 16: Identified muscle activity patterns in Hydra .............................................................................................. 44 Figure 17: Suction electrode recordings align recordings made with different indicators........................................... 62 Figure 18: Static fluorophore control........................................................................................................................... 63 iii ACKNOWLEDGEMENTS This work would not
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