PMC:1084331 / 47038-51048 JSONTXT

{"project":"2_test","denotations":[{"id":"15836427-11135642-84719690","span":{"begin":1357,"end":1359},"obj":"11135642"},{"id":"T56896","span":{"begin":1357,"end":1359},"obj":"11135642"}],"text":"Supporting Information\nFigure S1 Gene Expression Analysis in DRG Neurons of Er81EWS-Pea3 Mice\nAnalysis of TrkC expression by in situ hybridization (A and E), and Runx3 (red; B and F), Brn3A (red; C and G), Isl1 (red; D and H), p75 (red; I and M), TrkA (red; J and N), CGRP (red; K and O), Calretinin (CR; red; L and P), and LacZ (green; B–D and F–P) by immunohistochemistry on E16.5 lumbar DRG of Er81NLZ/+ (A–D and I–L) and Er81EWS-Pea3/− (E–H and M–P) embryos.\nScale bar: (A, B, E, F, L, and P), 70 μm; (C, D, G, H, I–K, and M–O), 75 μm.\n(4.9 MB CDR).\nClick here for additional data file.\nFigure S2 Ia Proprioceptive Afferents Make Functional Connections with Motor Neurons in Er81EWS-Pea3 Mutants\n(A and B) Representative traces from intracellular recordings measuring Ia afferent monosynaptic input to quadriceps motor neurons evoked by suprathreshold stimulation of the quadriceps nerve in wild-type (A) and Er81EWS-Pea3/− mutant (B) animals.\n(C) Average monosynaptic amplitudes (± standard error of the mean) from all recorded cells (wild-type, n = 11; mutant, n = 8).\n(20 KB CDR).\nClick here for additional data file.\nFigure S3 Generation of Mice Expressing EWS-Pea3 or mGFP in Early Post-Mitotic DRG Neurons\n(A) Top panel shows organization of the Tau genomic locus in the region targeted by homologous recombination in analogy to Tucker et al. [41]. Exons 1–3 are shown as light blue boxes, and the Tau start codon in exon 2 is indicated as ATG. The probe used to detect homologous recombination is shown as a grey box. Middle and bottom panels show Tau locus after homologous recombination to integrate targeting cassettes (green) into exon 2 with coincident elimination the endogenous Tau start codon. The integrated targeting cassettes allow for conditional expression of EWS-Pea3 and NLS-LacZ (NLZ) (middle) or mGFP and NLS-LacZ (bottom) upon Cre-recombinase-mediated activation. In the absence of Cre recombinase, a transcriptional stop sequence flanked by loxP sites inhibits expression of the respective transgenes from their start codons (ATG in grey).\n(B) Southern blot analysis of TauEWS-Pea3/+ and TaumGFP/+ genomic DNA to detect the mutant allele.\n(C) In the presence of Cre recombinase, the transcriptional stop sequence in the cassettes integrated into the Tau locus is removed. Expression of EWS-Pea3 and NLS-LacZ (top) or mGFP and NLS-LacZ (bottom) can now occur in neurons coincidently expressing Cre recombinase and Tau (indicated as ATG in green).\n(D–L) Expression of Isl1 (D, G, and J), EWS-Pea3 (E and H), GFP (K), or LacZ (F, I, and L), in E12 (D–I) or E13.5 (J–L) DRG neurons of wild-type (D–F), TauEWS-Pea3/+ Isl1Cre/+ (G–I), and TaumGFP/+ Isl1Cre/+ (J–L) embryos.\nScale bar: (D–F), 40 μm; (G–I), 35 μm; (J–K), 50 μm.\n(1.5 MB CDR).\nClick here for additional data file.\nFigure S4 Generation of PVCre Mice\n(A) Above is the organization of the PV genomic locus. Exons are schematically illustrated as light blue boxes, where exon 2 contains the start codon (ATG) and exon 5 contains the stop codon (STOP). Probe to screen for homologous recombination is shown as grey box. Below is a schematic diagram to show the PV locus after the integration of an IRES-Cre cassette (green) 3′ to the translational stop codon of PV using homologous recombination in ES cells.\n(B) Southern blot analysis of PVCre wild-type (+/+) and heterozygous (+/−) genomic DNA using the probe indicated in (A).\n(C and D) Expression of GFP (green) and LacZ (red; C) or PV (red; D) in P0 TaumGFP/+ PVCre/+ mice. Note that more than 90% of PV+ neurons coexpress GFP (D; data not shown).\nScale bar: (C and D), 50 μm.\n(2 MB CDR).\nClick here for additional data file.\nFigure S5 Gene Expression Analysis upon Precocious Induction of EWS-Pea3 in DRG Neurons\nImmunohistochemical analysis of PV (A and D), Calretinin (B and E), and Calbindin (C and F) expression on E16.5 lumbar DRG of wild-type (A–C) and TauEWS-Pea3/+ Isl1Cre/+ (D–F) embryos.\nScale bar: 80 μm.\n(950 KB CDR).\nClick here for additional data file."}