Generation of Conditional Knock-out Mice
A conditional RhoA allele, in which exon 3 and part of intron 3 are flanked by two loxP sites (RhoAfl/fl), was generated as depicted in . Prior to conducting experiments, the Neo cassette was removed by crossing the mice with Flippase-expressing mice. RhoAfl/fl mice were then crossed with RhoAfl/+;Nestin-Cre (RhoA het) mice to obtain RhoAfl/fl;Nestin-Cre (RhoA cKO) as well as RhoA het and RhoAfl/fl (control) littermates for experiments. Genotyping was performed by polymerase chain reaction (PCR).
Generation and characterization of mice containing a conditional allele ofRhoA.
All animal experiments were carried out in strict accordance with the recommendations provided by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (AN-4365). All efforts were made to minimize animal suffering.
The following purchased antibodies were used in our study: rabbit polyclonal antibodies anti-RhoA, anti-RhoB, and anti-RhoC (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-EphA4 receptor and rabbit polyclonal anti-ephrinB3 (Invitrogen, Camarillo, CA); anti-GAPDH antibody (Millipore, Temecula, CA); and the mouse monoclonal anti-acetylated tubulin antibody (Sigma, St. Louis, MO). For secondary antibodies, we used goat polyclonal antibodies labeled with horseradish peroxidase for Western blotting or with Cy3 or AlexaFluor 488 for immunostaining (Jackson ImmunoResearch Laboratories, West Grove, PA). Alexa 594-labeled phalloidin was purchased from Molecular Probes (Eugene, OR).
Protein Lysates and Western Blot Analysis
Primary neurons were lysed in NP-40 lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%; NP-40, 5% glycerol) containing protease inhibitors (complete mini cocktail, Roche, Mannheim, Germany) and phosphatase inhibitors for immunoblot analysis. Brain and spinal cord tissue were homogenized in NP-40 lysis buffer using a Dounce homogenizer. Protein amounts were estimated using Bradford reagent (Biorad, Carlsbad, CA). Cell lysates were separated using sodium dodecyl sulfate (SDS) –polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were incubated in blocking buffer [5% skimmed milk powder in 50 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween (TBST)] followed by an overnight incubation with primary antibody at 4°C. Horseradish-peroxidase-labeled secondary antibody binding was detected by enhanced chemiluminescence (ECL, Pierce, Rockford, IL). Autoradiography films (VWR International, Radnor, PA) were scanned and the bands were quantified using Image J software ().
Expression of Rho isoforms in the developing mouse brain and spinal cord.
A 40 cm long, 5 cm wide runway was lined with white paper at the bottom. Mice were trained to run down the runway in a straight line a day before the test. On the test day, the paws of the mice were dipped in non-toxic acrylic paint (forepaws red, hindpaws black) and mice were allowed to run down the runway. The footprints were analyzed for interstep distance (the distance between the placement of the right hindpaw or forepaw and the placement of the left hindpaw or forepaw, -a) and intrastep distance (the distance covered by the same hindpaw or forepaw, -b) to determine the degree of parallel movement of the limbs.
RhoA cKO mice display a rabbit-like hopping gait.
Anterograde Tracing and Analysis of the Corticospinal Tract
CST axons were labeled with biotinylated dextran amine (BDA; lysine fixable, MW 10,000; Molecular Probes, Eugene, OR) using a method previously described 
. Mice were anesthetized with isoflurane, positioned in a stereotaxic frame, and the right sensorimotor cortices exposed. A solution of BDA (10% in phosphate buffered saline (PBS), pH 7.4) was injected using a Nanoliter injector (World Precision Instruments, Sarasota, FL) into 8 sites (220 nl per site) in two rows of 4 sites in the right sensorimotor cortices that project to regions of the spinal cord that innervate the forelimbs and hindlimbs 
. The mice were sacrificed nine days after the BDA injection. The cervical regions of the spinal cord (C1–C7) were removed, postfixed for 6 hrs and then infiltrated with 21% sucrose in PBS for cryoprotection. Cross sections (40 µm) were cut on a cryostat and stored at –20°C until they were processed for histochemistry. The BDA labeled axons were visualized with avidin-biotin complexes reagent (Vector Laboratories, Burlingame, CA) and diaminobenzidine solution (0.7 mg/ml) containing 0.06% nickel chloride and 0.015% hydrogen peroxide. The number of axons was determined by an investigator who was unaware of the genotype of each animal. The quantity of axons was measured from the photomicrographs using NIH Image J software (NIH, Bethesda, MD) and the FeatureJ plug-in as described previously 
. The number of axons was measured as the number of pixels in the region (, MR) contralateral to the labeled CST. To avoid error resulting from variable BDA labeling efficiencies among mice, the number of pixels in a corresponding region (, ML) in the gray matter ipsilateral to the labeled CST was also measured. The degree of axons that innervated the spinal cord contralateral to the labeled CST was expressed as the ratio of the number of pixels in the region MR to the number of pixels in the region ML.
Aberrant midline crossing of corticospinal tract axons in RhoA cKO mice.
Measurement of Dorsal Column and Gray Matter
To compare the morphologic change of dorsal column, the height (vertical line in ) and width (horizontal line in ) of dorsal column at the cervical level were measured and the ratio of width to height was calculated. The space between bottom of dorsal column and the top of ventral column of gray matter at midline (vertical line in , middle panel) was also measured.
Abnormal morphology and incorrect innervation in the spinal cord of RhoA cKO mice.
Labeling of Spinal Interneurons
The spinal interneurons were traced as previously described 
with some modifications. Briefly, postnatal day 5 (P5) mice were deeply anaesthetized with isoflurane and decapitated. Spinal cords were dissected out in cold artificial cerebrospinal fluid (aCSF) (aCSF: 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2
, 25 mM NaHCO3
, 2 mM CaCl2
, 1 mM MgCl2
, 25 mM glucose) and then placed with the dorsal side facing up in a bath of aCSF bubbled with oxygen. Crystals of rhodamine dextran (Molecular probes, Eugene, OR) were applied to a unilateral cut at L4. The preparations were then incubated in oxygenated aCSF solution for 12–15 hr at room temperature. Spinal cords were then fixed in 4% PFA, infiltrated with 20% sucrose and cryosectioned into 50 µm thick sections. Sections were mounted in Fluoromount-G (Southern Biotech, Birmingham, AL) and imaged at L2 to detect axonal crossing using an epifluorescence microscope (Zeiss Axio). The experiment was repeated with four animals per genotype. The mean labeling intensity was quantified using NIH ImageJ software.
50 µm thick, cryostat-cut spinal cord sections at L2 level were subjected to immunohistochemistry for ephrinB3 staining. Briefly, the sections were blocked in 3% BSA with 0.1% Triton X-100 for 1 hr. The sections were then incubated overnight with anti-ephrinB3 antibody at 4°C. Alexa 488 anti-rabbit secondary antibody was added for 2 hr. After 3 washes with 1× PBS, the sections were mounted in Vectashield mounting medium with DAPI (Vector labs, Burlingame, CA).
Primary Cell Culture and Growth Cone Collapse Assay
Cortical neuron cultures were prepared from the developing motor corticies of P0 mouse pups or embryonic day 18 (E18) rat embryos and plated on coverslips coated with PDL/Laminin (BD Biosciences) in Neurobasal A supplemented with B27 supplement, 200 mM L-glutamine and penicillin/streptomycin. After 24 hrs, the medium was changed to Neurobasal A (Gibco, Grand island, NY) supplemented with astrocyte-conditioned medium (Ratio 3
1). To inhibit RhoA/ROCK signaling, rat cortical neurons were treated with 20 µM of the ROCK inhibitor Y-27632 (Tocris, Ellisville, MO) or vehicle for 1 hr. At 2 days in vitro (DIV2), the cortical neuron cultures were stimulated with 2 µg/ml of preclusted Fc (control) or ephrinB3-Fc (R&D systems, Inc, Minneapolis, MN) for 30 minutes. Fc and ephrinB3-Fc were preclusterd with an anti-Fc antibody (Jackson ImmunoResearch) in a 5
1 molar ratio at room temperature for 1 hr as previously described 
. The cells were fixed with 4% PFA for 15 min and processed for immunostaining to visualize F-actin and microtubules. The neurons were imaged and analyzed for growth cone collapse in a blinded manner. Growth cones with no filopodia or lamellipodia were scored as collapsed. Approximately 200 neurons were analyzed per condition for each experiment and the experiment was repeated 3 independent times.
Primary cortical neuronal cultures were fixed with 4% PFA for 15 min after the treatment at specified time points. The coverslips were washed five times with 1× PBS and blocked with the blocking solution (15% goat serum, 0.1% Triton ×100, 5% BSA in 1× PBS). Neurons were then incubated overnight with anti-acetylated tubulin antibody. After 3 washes the coverslips were incubated with Alexa 488-conjugated secondary antibody and Alexa 594-phalloidin (Molecular Probes, Eugene, OR). The cells were imaged using a Zeiss epifluorescence microscope.
All statistical parameters were calculated using KaleidaGraph software (Synergy Software, Reading, PA). Student’s t-test or analysis of variance (ANOVA) followed by Tukey's or Student-Newman-Keuls post-test were used to determine statistical significance. P values less than 0.001 were considered significantly different.