Technical Reference #702

702.

Shape Change Controls Supporting Cell Proliferation in Lesioned Mammalian Balance Epithelium Jason R. Meyers and Jeffrey T. Corwin, University of Virginia, The The Journal of Neuroscience, 27(702), (2007)
Link To Paper

Abstract:
Mature mammals are uniquely vulnerable to permanent auditory and vestibular deficits because the cell proliferation that producesreplacement hair cells in other vertebrates is limited in mammals. To investigate the cellular mechanisms responsible for t

Keywords:
hair cell; regeneration; lysophosphatidic acid; LPA; vestibular; wound healing; cytoskeleton

Materials & Methods:
Dissection of utricles. All animal experiments were performed according to protocols approved by the Animal Care and Use Committee at the University of Virginia. Swiss Webster mice were obtained from Taconic Farms (Germantown NY) and Charles River Laboratories (Wilmington MA) and Tg(GFPU)5Nagy mice were obtained from The Jackson Laboratory (Bar Harbor ME). Animals were killed by CO2 asphyxiation and decapitated. Labyrinths were removed aseptically from temporal bones in ice-cold DMEM/F-12 (Invitrogen Carlsbad CA) the utricles were isolated and the roof and the otoconia were removed. In vitro culture and lesioning. Embryonic utricles were adhered with the nerve side down on poly-L-lysine-coated glass-bottom dishes (Mat- Tek Ashland MA). Utricles from postnatal animals were adhered nerve side down in MatTek dishes treated with CellTak (2 l air-dried onto the glass and washed three times with medium; BD Biosciences San Jose CA). For time-lapse experiments embryonic utricles were adhered nerve side down to a 42mmround cover glass treated with CellTak. No difference in healing was observed between embryonic utricles attached via poly-L-lysine versus CellTak. Micropunches were fabricated by electrolytic etching of blunt 29 gauge stainless steel hypodermic needles (nominal internal diameter of 180 m; Hamilton Reno NV) in a solution of 34% sulfuric acid and 42% phosphoric acid at 4.5 V 500 mA. Rough edges were removed with 1 m grit polishing paper (3M Minneapolis MN) and etching and polishing were repeated until the tip had a sharp circular edge. The micropunches were pressed into utricles to make circular lesions in the hair cell epithelium and cells from within the lesion were removed with a sharpened tungsten needle. Lesioned utricles were then cultured in DMEM/F-12 with 5% FBS (HyClone Logan UT) 3 g/ml bromodeoxyuridine (BrdU; Sigma St. Louis MO) 0.25 g/ml Fungizone (Invitrogen) and 10 g/ml Ciprofloxacin (Bayer Berlin Germany) and fixed in Glyofixx (for immunocytochemistry) 4% paraformaldehyde in PBS (for phalloidin visualization of actin) or 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate (for electron microscopy). Utricles fixed for electron microscopy were made conductive by osmium– thiocarbohydrazide impregnation (Kelley et al. 1973; Malick and Wilson 1975). Time-lapse microscopy. Lesioned utricles were adhered to a 42-mmdiameter cover glass with CellTak (BD Biosciences) for time-lapse microscopy. The cover glass was sealed into a POC-R imaging chamber (LaCon Staig Germany) containing DMEM/F-12 with 5% FBS 3 g/ml BrdU 0.25 g/ml Fungizone and 10 g/ml Ciprofloxacin. The chamber was placed on an Axiovert 200M microscope (Zeiss Oberkochen Germany) with the epithelia oriented downward for imaging. A microscope stage incubator (PeCon Erbach-Bach Germany) was maintained at 5% CO2 and 37°C for the duration of the experiment. A motorized stage that was under MetaMorph software control (Molecular Devices Sunnyvale CA) allowed sequential imaging of responses occurring concurrently in 2–12 utricles throughout each time-lapse recording. Every 10–30 min images were recorded at each of 10 focal levels extending through the depth of the sensory epithelium for each utricle in the chamber. For time-lapse tests of pharmacologic agents utricles were divided into equal groups and preincubated for 30 min at 37°C in either the control medium or medium supplemented with the drug. Then all of the utricles were lesioned with a micropunch and the utricles in each pool (control and experimental) were adhered to a single 12-mm-diameter cover glass coated with CellTak. The control and experimental cover glasses were each fitted with a custom-made ring of 250- m-thick silicone gasket material (McMaster-Carr Atlanta GA) and either control medium or medium containing a pharmacological agent was added to the small well formed by the gasket. The two gasketed cover glasses were then inverted onto a 42mmcircular cover glass creating two individually sealed compartments within the same POC-R imaging chamber. To block actin polymerization utricles were cultured in 1 M cytochalasin D or 1 M cytochalasin B (Sigma) and controls were cultured in 0.1% DMSO as a vehicle control. To inhibit Rho-mediated kinases utricles were cultured in 50 M (R)-( )-trans-N-(4-pyridyl)-4-(1- aminoethyl)-cyclohexanecarboxamide (Y-27632; EMD Biosciences La Jolla CA). To inhibit myosin light chain kinase (MLCK) utricles were cultured in 1 or 20 M 1-(5-iodonaphthalene-1-sulfonyl)homopiperazine (ML-7; EMD Biosciences). To washout media containing drugs the gasketed coverslips were lifted out of the POC-R chamber and the cover glass was washed in four changes of fresh drug-free culture medium over 20 min before the chambers were reassembled with fresh culture medium without drugs for continued imaging. Time-lapse of green fluorescent protein-expressing utricles. To visualize changes in individual cell shapes time-lapse recordings were made using utricles removed at embryonic day 18 (E18) (n 11) or on the day of birth [postnatal day 0 (P0)] (n 12) from transgenic mice that expressed green fluorescent protein (GFP) under control of the -actin promoter [Tg(GFPU)5Nagy] (Hadjantonakis et al. 1998). Variations in the levels of GFP fluorescence among the cells in the sensory epithelium allowed individual cells to be tracked during the course of the recording (see Fig. 2). Images over 10 focal planes each 10 m apart were acquired every hour for 24 h using a Zeiss 510 confocalLSMwith an incubation chamber as described above. The positions of individual bright or dim cells were then tracked via automated object tracking using MetaMorph software in which the center of each cell was determined in each frame based on an object-specific template defined by differences in signal intensity and shape. The template was updated with each frame to adjust for changes during the time-lapse recordings. LPA stimulation of mature utricles. To exogenously stimulate Rho 24 mature utricles were cultured in the presence of 10 M lysophosphatidic acid (Avanti Polar Lipids Alabaster AL). A 1 mM stock of LPA was made in 3% fatty-acid-free bovine serum albumin (FAF-BSA; Sigma) in water; 21 controls were cultured in FAF-BSA without LPA. LPA is released from platelets in the standard harvesting of serum (Eichholtz et al. 1993) so we sought to reduce potential variability by maintaining both the experimental and control cultures in charcoal– dextran-filtered FBS (Hy- Clone). Half of the media was changed every 12 h. LPA stimulation of delaminated sensory epithelia. Cultures of isolated utricular epithelium were obtained as by Montcouquiol and Corwin (2001b). Briefly 2-week-old utricles were incubated in thermolysin (0.5 mg/ml; Sigma) for 45 min at 37°C and the sensory epithelium was removed and plated on glass-bottom dishes (MatTek) coated with poly-Llysine. Sensory epithelia were cultured in DMEM/F-12 with 5% charcoal– dextran-filtered FBS containing 10 M LPA. Images of the epithelia were obtained after plating and every 24 h for 3 d. Immunohistochemistry. Cultures for phalloidin labeling were fixed in 4% paraformaldehyde in PBS overnight at 4°C washed in PBS containing 0.2% Triton X-100 (PBST) three times over 15 min followed by a 15 min incubation in AlexaFluor-labeled phalloidin (5 U/ml; Invitrogen) in PBST and then washed three times over 15 min in PBST.

Microscopic Technique
Electron Microscopy, Time-Lapse Microscopy

Cell Type(s)
Hair, epithelium