title: Methods for imaging individual cilia in living echinoid embryos.
abstract: The embryos of echinoids (sea urchins and sand dollars) serve as excellent models for studying cilia differentiation and stages of the cilia life cycle including ciliogenic initiation, growth, maintenance, and retraction. Early in echinoid development, uniform motile cilia form on all cells simultaneously but then rapidly differentiate into multiple cilia types that differ in morphology, motility, and signaling sensitivity. Metal ion treatments that shift germ layer boundaries and thereby "animalize" or "vegetalize" embryos can be used to enrich for low-abundance cilia types rendering those specialized cilia and the differentiation processes they exhibit much easier to study. The experimental advantages of having robust cilia growth and differentiation is tempered by the challenge of restraining ciliated embryos well enough to view the process of ciliogenesis live. We have developed four observation chambers as modifications of the Kiehart chamber for long-term light microscopic imaging of ciliated echinoid embryos. One of these systems employs paramagnetic beads to render ciliated larvae magnetic so they can be gently and reversibly trapped directly under the objective lens. With this magnetic trapping system, the larva can be positioned and repositioned until they achieve the orientation with the clearest view of any cilia of interest. These methods of gentle embryo restraint allow normal embryo development and the normal ciliogenic cycle and ciliary differentiation processes to continue in direct view. Sequential image series can then be collected and analyzed to quantitatively study the wide spectrum of cilia behaviors and properties that arise in developing echinoid embryos.
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Morris
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Hans W
authors.initials: HW
authors.lastname: Pope
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Adam N
authors.initials: AN
authors.lastname: Sholi
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Leah M
authors.initials: LM
authors.lastname: Williams
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA.
authors.forename: Chelsea R
authors.initials: CR
authors.lastname: Ettinger
authors.affiliation: Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA; Honors College, University of Maine, Orono, ME, USA.
authors.forename: Gwendolyn M
authors.initials: GM
authors.lastname: Beacham
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Shintaku
authors.affiliation: Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA; Department of Biology, Bates College, Lewiston, ME, USA.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Abbott
authors.affiliation: Department of Biology, Wheaton College, Norton, MA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Elyse M
authors.initials: EM
authors.lastname: Doherty
mesh_terms: D000818:Animals; D002923:Cilia / Q000502:physiology* / Q000648:ultrastructure*; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000502:physiology; D047108:Embryonic Development; D007091:Image Processing, Computer-Assisted; D007814:Larva / Q000254:growth & development*; D058185:Magnetite Nanoparticles; D061848:Optical Imaging / Q000379:methods*; D012617:Sea Urchins; D046509:Tissue Culture Techniques
chemical_list: D058185:Magnetite Nanoparticles
keywords: Animalize; Embryo; Imaging; Kiehart chamber; Ledge chamber; Magnetic; Sand dollar; Sea urchin; Vegetalize; Wedge chamber