Materials and Methods

Generation of double KOs.
We crossed Bax KO females on a C57BL/6 background (Jackson Laboratory, Bar Harbor, Maine, United States) to NT-3 heterozygote males on a 129 Sv background to generate double heterozygote animals. Progeny was genotyped with PCR, and animals heterozygous for both genes were bred to obtain the double KOs. Primers used for the Bax locus were R661, GTT GAC CAG AGT GGC GTA GG; R662, CCG CTT CCA TTG CTC AGC GG; and R663, GAG CTG ATC AGA ACC ATC ATG. Primers used for the NT-3 locus were NT3A, CGT GGT GAG GTT CTA TTG GCT AC; NT3B, CAG AGC ACC CTG CCC AAA GCA GAG; NT3R, CCT TGA CAA TAC TGA ATG CC; and NEOF, GGG AAC TTC CTG ACT AGG. WT, Bax KO, and NT-3 KO littermates were used as controls. A total of 11 Bax/NT-3 double KO mice were analyzed, of these nine were P0 pups. The p75 colony on a 129 S1 background was received from Jackson Laboratory. We used tail DNA to genotype animals using the primers IMR0013, CTT GGG TGG AGA GGC TAT TC; IMR0014, AGG TGA GAT GAC AGG AGA TC (generic neo primers); IMR0710, TGT TAC GTT CTC TGA CGT GGT GAG; and IMR0711, TCA GCC CAG GGT GTG CAC TC (p75 locus). For embryonic experiments, day of plug positivity was considered E0. All of the protocols used in this study were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guidelines for use of experimental animals.

TrkA/TrkC immunohistochemistry.
Frozen spinal cord sections (10 μm thick) were blocked and incubated in a cocktail of rabbit anti-TrkA and goat anti-TrkC antibodies (gift of Dr. Reichardt; Huang et al. 1999), followed by a cocktail of CY3 conjugated donkey anti-rabbit and FITC conjugated donkey anti-goat antibodies (Chemicon, Temecula, California, United States) in the presence of 0.3% TritonX-100 and 10% normal donkey serum. For PV immunohistochemistry, sections were reacted with monoclonal mouse anti-PV antibody (Sigma, St. Louis, Missouri, United States), and developed by Vector fluorescein mouse on mouse kit (Vector Laboratories, Burlingame, California, United States). For quantification purposes, TrkA- and TrkC-labeled cells from four different DRGs of each genotype and age studied were counted, and ratios plotted.

Muscle spindle detection.
Gastrocnemius muscle was dissected out in P0 pups and sectioned longitudinally, or, in some cases, the whole leg at the level of gastrocnemius muscle with tibial nerve was sectioned in cross section. Sections were incubated with monoclonal S46 antibody (gift of Dr. Stockdale) reactive to the spindle-specific slow-tonic myosin heavy chain isoform and developed by Vector mouse on mouse kit as described above, followed by rabbit anti-NF-M antibody (Chemicon) and CY3 conjugated goat anti-rabbit antibody (Chemicon) in a sequential double-labeling protocol. In another method, DiI crystals were placed in DRGs of E17 embryos from different genotypes (n = 2), and the gastrocnemius muscle was isolated and sectioned into 40-μm-thick slices on a vibratome.

DiI labeling.
Spinal cords were dissected out with the DRGs attached, motor root was cut to prevent backfilling of motor neurons, and crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, Oregon, United States) were inserted in DRGs. Labeled spinal cords were incubated at 37 °C for 8 d and sectioned on a vibrotome at 100 μm. Sections were observed under the fluorescent microscope and photographed, and images were transferred to Adobe Photoshop, inverted, and adjusted for brightness and contrast. Photoconverted in 0.15% DAB (3,3′-diaminobenzidine; Sigma) in 0.1M Tris buffer (pH 8.2).

In vitro co-culture assay.
DRG explants were derived from Swiss Webster, Bax null, and p75 null mouse embryos (E13). For collagen matrix assays, DRGs were dissected out under a stereomicroscope using tungsten needles. Collagen matrix was prepared with 430 μl of collagen (3 mg/ml dissolved in 0.1M acetic acid, Sigma), 50 μl of 10X DMEM medium, and 2.5 μl of 0.8 M NaCa2, and the pH was adjusted to 7.5. Individual ganglion explants were placed in 24-well plates and covered with freshly prepared collagen. Sepharose beads with an average diameter of 150 μm were used. Beads were washed twice with PBS, air dried, and loaded with 10, 20, 50, or 100 ng/μl NT-3 (Collaborative Research, Chemicon) at 4 °C overnight with constant shaking. For negative control, beads were loaded with BSA (10–100 ng/μl) or PBS. Either a single neurotrophin-loaded bead or a single BSA (or PBS)–loaded bead was implanted about 200–1,200 μm away from the ganglion explant. Collagen-embedded cultures were then placed at 33 °C for 15 min for the matrix to harden. Serum-free culture medium was then added to each well. In cultures with WT DRG and control beads, 10% serum was added to ensure viability of the explants. TrkC-Fc (Regeneron Pharmaceuticals, Tarrytown, New York, United States) was added (20 μg/ml) into the culture medium.

Explant co-cultures
Spinal cords with attached DRGs were dissected out from Swiss Webster mouse embryos at E13, and sectioned into 300-μm-thick slices. Tissue slices were placed on Millicell Tissue Culture Inserts (Millipore, Billrica, Massachusetts, United States). NT-3-loaded (n = 15) or PBS-loaded (n = 6) sepharose beads were prepared as described above, and placed in the midline at mid-spinal cord level. Inserts were placed in six-well plates with serum-free medium at the bottom of the wells, and kept at 33 °C for 3 d in the presence of 5% CO2. Cultures were then fixed with 4% paraformaldehyde in PBS, and small crystals of DiI were placed into the DRGs. Samples were incubated in a 37-°C incubator, allowing the dye to diffuse, and photographed under a Nikon (Tokyo, Japan) inverted epifluoresence microscope.