Technical Reference #269

269.

Regeneration in avian hair cell epithelia: identification of intracellular signals required for S-phase entry Mark C. Witte; Mireille Montcouquiol and Jeffrey T. Corwin, University of Virginia, European Journal of Neuroscience, 14(269), (2001)
Link To Paper

Abstract:
Balance epithelia in birds closely resemble their mammalian counterparts but their cells turnover rapidly and they quicklyregenerate hair cells leading to functional recovery from damage that would be permanent for a mammal. We isolated andcultured

Materials & Methods:
Epithelial cell cultures Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals using a protocol approved and supervised by the University of Virginia Animal Care Advisory Committee. For this investigation 72 white leghorn chickens (Gallus domesticus) that were 1±7 days old were anaesthetized with CO2 and decapitated. The skin and the mandibles were removed and the heads were immersed in chilled 70% ethanol for 15 min. Then the lateral squamous portion of the temporal bone was removed with a sterile razor blade. The utricles were dissected from the membranous labyrinth and transferred to a chilled solution of medium 199 with Hanks' salts (M199-H) and the utricle's roof and otolithic membrane were removed. Then the utricles were incubated in thermolysin (protease type X Sigma St Louis MO USA) at 0.5 mg/mL in Hanks' balanced salt solution containing 25 mM HEPES at pH 7.4 in a 5% CO2 incubator at 37 °C for 45 min (Saffer et al. 1996). The utricles were then transferred to M199-H which contained 10% fetal bovine serum (FBS Hyclone Laboratories Logan UT USA) added to retard further enzymatic digestion. The sensory epithelium was carefully dissected free from the underlying connective tissue using ®ne forceps. The epithelial edges were trimmed away with a diamond microscalpel and discarded to remove all traces of the surrounding nonsensory epithelium. The remaining sheet of pure sensory epithelium was cut into pieces that measured » 0.25 mm2 each. Eight to 12 pieces were transferred in M199 with Earle's salts (M199- E) and 10% FBS to the glass-bottom well of a Mat-Tek culture dish that had been coated by overnight exposure to 0.1 mg/mL ®bronectin (Sigma). A needle was used to orientate the pieces so that their hair bundles were up then they were allowed to attach and grow undisturbed for 96 h in a 5% CO2 atmosphere at 37 °C. After the 96-h growth period the standard tests began with a 1-h preincubation with the inhibitor in M199-E supplemented with 50 mg/mL holo-transferrin 5 ng/mL sodium selenite and 1 ng/mL bovine insulin (Sigma). Then the medium was replaced with M199-E containing 10% FBS the inhibitor and 5-bromo-2-deoxyuridine (BrdU) at 3 mg/mL to label DNA during the synthesis (S-phase) of the cell cycle. After 8 h of incubation in the BrdU-containing medium the cultures were ®xed with 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS) at pH 7.4. The protocol allowed inhibitors to act on their target enzymes in a relatively nutrientdepleted de®ned medium for 1 h prior to the test of the inhibitor's effect in a medium that contained 10% FBS to again prompt cell proliferation. This experimental time-line was modi®ed when FK506 was used to test the speci®city of the inhibition by rapamycin. In that experiment the cultures were preincubated with FK506 in the de®ned medium for 1 h prior to the addition of rapamycin. This step was followed by an additional 1-h preincubation in de®ned medium containing both FK506 and rapamycin. Then the cultures were incubated for 8 h in medium containing FK506 and rapamycin with 3 mg/mL BrdU and 10% FBS. One further modi®cation of the experimental time-line was employed when a three-inhibitor combination (PD 98059 rapamycin and LY294002) was used in a prolonged preincubation protocol that preceded the addition of BrdU. In this experiment an additional 12-h preincubation period was added prior to the standard 1-h preincubation in de®ned medium. In order to avoid deleterious effects caused by abrupt and prolonged withdrawal of the nutrient growth medium the medium used for the 12-h preincubation contained 10% FBS. The remainder of the procedure followed the standard experimental timeline. The inhibitors and the concentrations used were LY294002 at 30 mM wortmannin at 25 nM rapamycin at 20 nM FK506 at 12 mM calphostin C at 0.5 mM bisindolylmaleimide I (BIM) at 1 mM AND 2 mM GoÈ6983 at 100 nM PD98059 at 50 mM AND 100 mM U0126 at 10 mM AND 20 mM and apigenin at 13 mM AND 39 mM. The concentrations chosen for the inhibitors used were based on previous studies and in particular a study of utricle epithelia from rats that used a similar culture technique (Montcouquiol & Corwin 2001). The FK506 was from Fujisawa (Deer®eld IL USA). The PD98059 wasfrom New England Biolabs (Beverly MA USA). All other inhibitors were purchased from Calbiochem (La Jolla CA USA). During each experiment 10±15 control cultures were run along with the experiments. The control data from all the experiments were pooled and are presented in several of the histograms from the individual experiments.

Microscopic Technique
Differential interference contrast microscopy (DIC)

Cell Type(s)
sensory epithelium