used in this study represent seven developmental stages (S28, S31, S37, S42, S45, S47, S50) that span a period between 1 and 15 days post-fertilization at 23°C. X. laevis
were purchased from a commercial supplier as newly fertilized eggs or larva (NASCO, Fort Atkinson, WI) and maintained in aerated aquaria at 22-24°C under full spectrum lights. Ten to fifteen larvae, each from a separate breeding, were used to acquire data for each stage. Morphological features, as viewed under a dissecting microscope, were used to determine the developmental stage of the specimens according to the descriptions of Neiuwkoop and Faber (1967)
. Criteria used to stage animals included characteristics such as the amount of pigmentation in the ventral portion of the tail region, the extent of eye and tentacle formation, the number of intestinal revolutions, the relative development of the hindbud, and the length of the body and hindlimb. The stages identified by Neiuwkoop and Faber (1967)
can be grouped into categories that represent embryonic (S28,S31), hatchling (S37, S42), and early larval (S45, S47, S50) developmental periods (McDiarmid and Altig, 1999
; Bever et al., 2003
). After staging, larvae were euthanized in buffered (pH 7.4) 0.25% 3-aminobenzoic acid ethyl ester methanesulfonate salt (Sigma A-5040) for 30 minutes prior to further tissue processing. Experimental procedures were approved by the Institutional Animal Care and Use Committee of New Mexico State University. Following euthanasia, larvae were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS; 140 mM NaCl, 2 mM KCl, 10 mM NaHPO4
, 1 mM KH2
) for 2 hours at room temperature (22-26 °C) then rinsed in PBS (3 times, 5 minutes each). Specimens were placed in 25 mM EDTA for 30 minutes at room temperature, rinsed in PBS (3 times, 5 minutes each) and processed for resin or paraffin embedding.
Figures illustrating the anatomical changes that occur during development were prepared using images of resin embedded sections. Specimens for resin embedded sections were dehydrated in 70% EtOH for 15 minutes, followed by 95% EtOH (2 times, 10 minutes each), and 100% EtOH (2 times, 10 minutes each). Specimens were submerged in 100% propylene oxide (PPO) for 5 minutes prior to infiltration with 50% resin in PPO (v/v) for 1 hour, and 75% resin in PPO (v/v) for 2-3 hours. Tissue then was placed in 100% ARALDITE 502 resin (EMS 10900) for 6-8 hours and cured at 50 °C overnight (18-22 hrs). Transverse 3-5 μm sections (perpendicular to the plane of the animal's body anterior to posterior) were prepared with a LKB ultramicrotome. Sections were mounted on Superfrost Plus slides (MJ Research), stained with 1% toluidine blue/1% borax (w/v), rinsed in tap water, then coverslipped using Histomount mounting medium (National Diagnostics HS-103). Staining times varied with animal stage.
Paraffin sections were used to assess animal variation in morphology and for dimensional measurements of the developing X. laevis inner ear. Specimens for paraffin embedded sections were dehydrated in a graded EtOH/H2O (v/v) series (25%, 50%, 75%, 100%) for 10-15 minutes in each solution, then infiltrated with a graded xylene/EtOH (v/v) series (25%, 50%, 75%, 100%) for 10 minutes in each solution. Specimens were infiltrated with a graded paraplast/xylene (v/v) mixture (25%, 50%, 75%, 100%) for 20 – 25 minutes each at 55°C and embedded in 100% Paraplast Plus (Tyco Healthcare/Kendall 8889-502004). Transverse 10 μm sections were prepared with a Zeiss microtome and mounted on Superfrost Plus slides, then placed in an oven overnight at 55°C prior to staining. Sections were deparaffinized in 100% xylene for 7-10 minutes, rehydrated through a series of decreasing EtOH alcohol solutions for 5 minutes each (100%, 75%, 50%, 25%), then placed in water. Sections were stained with Harris hematoxylin (Sigma HHS-16), rinsed in tap water, differentiated in an acid alcohol solution, blued in Scott's solution, and stained with alcoholic eosin Y (Sigma E4382) in 95% ethanol. Slides were dehydrated in 95% and 100% EtOH/H2O (2 times, 3 minutes each), cleared in Histoclear-II (National Diagnostics HS-202) for 5 minutes, then coverslipped using Histomount mounting medium.
Specimens were prepared for fluorescence microscopy and imaging of stereociliary bundles from specimens that were staged as above, then euthanized and fixed in 4% paraformaldehyde/PBS for 2 hours. For cryosections, specimens were rinsed in PBS for 10 minutes (2 times), embedded in 5% sucrose/5% agar (Becton Dickinson, 214010), and cryoprotected overnight at 4°C in 30% sucrose/0.1% sodium azide in PBS. The tissue was sectioned to 20 μm using a Leica CM 3050 cryotome, then stored at –20°C. Slides were placed at 37°C for 30 minutes before labelling with fluorescent probes. With our labelling protocols, the stereociliary bundle morphology in cryosections was superior to that in paraffin sections. Whole mounts were prepared for fluorescence microscopy from the inner ears of euthanized stage 47 and stage 50 specimens. Otic capsules (S47) and dissected inner ears (S50) were fixed in 4% paraformaldehyde/PBS for 2 hours, then immersed in 25 mM EDTA overnight at 4°C. The tissue was rinsed twice for 10 minutes in PBS prior to fluorescent staining. Sections and whole mounts were permeabilized and exposed to the F-actin label, Alexa 488 phalloidin (Molecular Probes A-12379), and the nucleic acid label, propidium iodide (Molecular Probes P-3566), according to the manufacturer's protocols, and without RNAase treatment. Cryosectioned tissue and whole mounts were coverslipped using SlowFade Light Antifade mounting medium (Molecular Probes S-7461).
Digitized images from paraffin and resin embedded sections were captured using a Zeiss Axioplan light microscope and WinView 32 image acquisition system (Princeton Instruments). A BioRad 1024 laser scanning confocal microscope configured with the E1 and T2 filter blocks was used to acquire digitized images from cryosectioned tissue and inner ear wholemounts labelled with fluorescent probes. Alexa 488 fluorescence was detected using a 522DF35 emission filter and the 488 Argon/Krypton laser line. Propidium iodide fluorescence was detected using a E585LP emission filter and excitation with the 568 Argon/Krypton laser line. Gain, aperture, and laser intensity settings were adjusted to minimize photobleaching. Autofluorescence was not detected from unlabelled inner ear tissue at the settings used for image capture from fluorophore-labelled tissue. Images were processed using BioRad Lasersharp 2000 software.