In situ hybridization and immunohistochemistry
For in situ hybridization analysis, cryostat sections were hybridized using digoxigenin-labeled probes [45] directed against mouse TrkA or TrkB, or rat TrkC (gift from L. F. Parada). Antibodies used in this study were as follows: rabbit anti-Er81 [14], rabbit anti-Pea3 [14], rabbit anti-PV [14], rabbit anti-eGFP (Molecular Probes, Eugene, Oregon, United States), rabbit anti-Calbindin, rabbit anti-Calretinin (Swant, Bellinzona, Switzerland), rabbit anti-CGRP (Chemicon, Temecula, California, United States), rabbit anti-vGlut1 (Synaptic Systems, Goettingen, Germany), rabbit anti-Brn3a (gift from E. Turner), rabbit anti-TrkA and -p75 (gift from L. F. Reichardt), rabbit anti-Runx3 (Kramer and Arber, unpublished reagent), rabbit anti-Rhodamine (Molecular Probes), mouse anti-neurofilament (American Type Culture Collection, Manassas, Virginia, United States), sheep anti-eGFP (Biogenesis, Poole, United Kingdom), goat anti-LacZ [14], goat anti-TrkC (gift from L. F. Reichardt), and guinea pig anti-Isl1 [14]. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to detect apoptotic cells in E13.5 DRG on cryostat sections was performed as described by the manufacturer (Roche, Basel, Switzerland). Quantitative analysis of TUNEL+ DRG cells was performed essentially as described [27]. BrdU pulse-chase experiments and LacZ wholemount stainings were performed as previously described [46]. For anterograde tracing experiments to visualize projections of sensory neurons, rhodamine-conjugated dextran (Molecular Probes) was injected into single lumbar (L3) DRG at E13.5 or applied to whole lumbar dorsal roots (L3) at postnatal day (P) 5 using glass capillaries. After injection, animals were incubated for 2–3 h (E13.5) or overnight (P5). Cryostat sections were processed for immunohistochemistry as described [14] using fluorophore-conjugated secondary antibodies (1:1,000, Molecular Probes). Images were collected on an Olympus (Tokyo, Japan) confocal microscope. Images from in situ hybridization experiments were collected with an RT-SPOT camera (Diagnostic Instruments, Sterling Heights, Michigan, United States), and Corel (Eden Prairie, Minnesota, United States) Photo Paint 10.0 was used for digital processing of images.