Supporting Information
Figure S1  Gene Expression Analysis in DRG Neurons of Er81EWS-Pea3 Mice
Analysis 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.
Scale bar: (A, B, E, F, L, and P), 70 μm; (C, D, G, H, I–K, and M–O), 75 μm.
(4.9 MB CDR).
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Figure S2  Ia Proprioceptive Afferents Make Functional Connections with Motor Neurons in Er81EWS-Pea3 Mutants
(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.
(C) Average monosynaptic amplitudes (± standard error of the mean) from all recorded cells (wild-type, n = 11; mutant, n = 8).
(20 KB CDR).
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Figure S3  Generation of Mice Expressing EWS-Pea3 or mGFP in Early Post-Mitotic DRG Neurons
(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).
(B) Southern blot analysis of TauEWS-Pea3/+ and TaumGFP/+ genomic DNA to detect the mutant allele.
(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).
(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.
Scale bar: (D–F), 40 μm; (G–I), 35 μm; (J–K), 50 μm.
(1.5 MB CDR).
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Figure S4  Generation of PVCre Mice
(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.
(B) Southern blot analysis of PVCre wild-type (+/+) and heterozygous (+/−) genomic DNA using the probe indicated in (A).
(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).
Scale bar: (C and D), 50 μm.
(2 MB CDR).
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Figure S5  Gene Expression Analysis upon Precocious Induction of EWS-Pea3 in DRG Neurons
Immunohistochemical 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.
Scale bar: 80 μm.
(950 KB CDR).
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