Materials and Methods Generation of transgenic mice and mouse genetics Er81EWS-Pea3 mice were generated following a strategy similar to that described for the generation of Er81NLZ mice [14]. In brief, a targeting vector with a cDNA coding for EWS-Pea3 was inserted in frame with the endogenous start ATG into exon 2 of the Er81 genomic locus and used for homologous recombination in W95 ES cells. EWS-Pea3 represents a fusion gene between the amino terminal of EWS and the ETS domain of Pea3 [20]. The primer pair used to specifically detect the Er81EWS-Pea3 allele was 5′- CAGCCACTGCACCTACAAGAC-3′ and 5′- CTTCCTGCTTGATGTCTCCTTC-3′. For the generation of TaumGFP and TauEWS-Pea3 mice, lox-STOP-lox-mGFP-IRES-NLS-LacZ-pA and lox-STOP-lox-EWS-Pea3-IRES-NLS-LacZ-pA targeting cassettes were integrated into exon 2 of the Tau genomic locus (the endogenous start ATG was removed in the targeting vectors; details available upon request). mGFP was provided by P. Caroni [25]. ES cell recombinants were screened by Southern blot analysis using the probe in the 5′ region as described previously [41]. Frequency of recombination in 129/Ola ES cells was approximately 1/3 for both Tau constructs. For the generation of PVCre mice, mouse genomic clones were obtained by screening a 129SV/J genomic library (Incyte, Wilmington, Delaware, United States). For details on the genomic structure of the mouse PV locus see [42]. An IRES-Cre-pA targeting cassette [33] was integrated into the 3′ UTR of exon 5, and ES cell recombinants were screened with a 5′ probe (oligos, 5′- GAGATGACCCAGCCAGGATGCCTC-3′ and 5′- CTGACCACTCTCGCTCCGGTGTCC-3′; genomic DNA, HindIII digest). The frequency of recombination in 129/Ola ES cells was approximately 1/20. Recombinant clones were aggregated with morula stage embryos to generate chimeric founder mice that transmitted the mutant alleles. In all experiments performed in this study, animals were of mixed genetic background (129/Ola and C57Bl6). Thy1spGFP transgenic mice were generated in analogy to De Paola et al. [25], and for these experiments a strain of mice with early embryonic expression was selected. Isl1Cre and Hb9Cre mouse strains have been described [33,43] and Bax+/− animals were from Jackson Laboratory (Bar Harbor, Maine, United States) [27]. Timed pregnancies were set up to generate embryos of different developmental stages with all genotypes described throughout the study. Transcriptional transactivation assays The following plasmids were used for transcriptional transactivation assays: pRc/RSV (Invitrogen, Carlsbad, California, United States), pRc/RSV-Er81, pRc/RSV-EWS-Pea3, pTP-5xETS, and pTP-5xETSmut. pRc/RSV-Er81 and pRc/RSV-EWS-Pea3 were obtained by insertion of the cDNAs for Er81 or EWS-Pea3 (gift from J. A. Hassell) into pRc/RSV. pTP-5xETS was constructed by inserting a cassette of five repetitive copies of high-affinity Pea3 binding sites (5′- GCCGGAAGC-3′) [18,19] into a modified version of pTK-Luc. pTP-5xETSmut was generated as pTP-5xETS but using a mutated complement of the Pea3 binding sites (5′- GCCTATGGC-3′). A control plasmid to normalize for transfection efficiency (placZ) and pTK-Luc were a gift from D. Kressler. COS-7 cells were co-transfected with 1–1.2 μg of total DNA including one of the effector plasmids pRc/RSV-empty, pRc/RSV-Er81, or pRc/RSV-EWS-Pea3; one of the reporter plasmids pTP-5xETS or pTP-5xETSmut; and placZ. Cells were harvested after 25 h and processed for assays to determine luciferase and LacZ activity as described previously [44]. Luciferase values normalized to LacZ activity are referred to as luciferase units. 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. In vitro cultures of DRG Individual lumbar DRG were dissected from E13.5 or E14.5 embryos and placed on Matrigel (BD Biosciences, San Jose, California, United States) coated coverslips in DMEM/F12 (Gibco, San Diego, California, United States), 2 mM L-Gln (Gibco), N2 (Gibco), and 1 mg/ml BSA (Sigma, St. Louis, Missouri, United States) without neurotrophins, or supplemented with either NGF (100 ng/ml, Gibco) or NT-3 (20 ng/ml, Sigma). DRG explants (n ≥ 20 for each condition) were cultured for 48 h, processed for immunocytochemistry, and analyzed using confocal microscopy. Western blot analysis Lumbar DRG from E16.5 embryos were isolated, mechanically disrupted, homogenized using glass beads (Sigma), and lysed in standard lysis buffer supplemented with protease and phosphatase inhibitors as described [47]. Protein extracts were resolved by SDS-PAGE, and immunoblotting was performed using antibodies against Akt, p-Akt (Ser473), CREB, p-CREB (Ser133), Bax, Bcl-xl (Cell Signaling Technology, Beverly, Massachusetts, United States), and Bcl2 (BD Pharmingen, San Diego, California, United States). For quantification, films (X-OMAT AR, Eastman Kodak, Rochester, New York, United States) were scanned and densitometry was performed using IMAGEQUANT 5.2 (Molecular Dynamics, Amersham, Uppsala, Sweden). Electrophysiology Electrophysiological analysis was performed as previously described [48]. Briefly, intracellular recordings from identified quadriceps motor neurons were made using sharp electrodes (75–120 MΩ, 3M KCl). Average responses (10–20 trials) from suprathreshold nerve stimulation (1.5 times the strength that evokes maximal monosynaptic response) of the quadriceps nerve were acquired with LTP software [49]. Only cells with stable resting potentials more negative than −50 mV were considered for analysis. Monosynaptic amplitudes were determined offline using custom routines in the Matlab environment (The Mathworks, Natick, Massachusetts, United States) as previously described [48].