Precocious Expression of EWS-Pea3 in DRG Neurons Leads to Axonal Projection Defects
To address the consequences of precocious ETS signaling for proprioceptive afferent differentiation, we next expressed EWS-Pea3 in DRG neurons as soon as they became post-mitotic. We used a binary mouse genetic system based on Cre-recombinase-mediated excision of a transcriptional stop cassette flanked by loxP sites. Targeting cassettes were integrated into the Tau locus to generate two strains of mice conditionally expressing either EWS-Pea3 or a membrane-targeted green fluorescent protein (mGFP) to trace axonal projections of DRG neurons (Figure S3; [25]). Embryos positive for either Isl1Cre/+ and TauEWS-Pea3/+ or Isl1Cre/+ and TaumGFP/+ alleles showed efficient activation of the silent Tau allele in 95% or more of all DRG neurons, including proprioceptive afferents, at all segmental levels (Figure S3).
We first assessed the influence of EWS-Pea3 expression in early post-mitotic DRG neurons on the establishment of afferent projections into the spinal cord using the TaumGFP/+ allele or a Thy1-promoter-driven synaptophysin green fluorescent protein (spGFP) with an expression profile restricted to DRG sensory neurons at embryonic day (E) 13.5 (Thy1spGFP; [25]) (Figure 3). In contrast to wild-type proprioceptive afferent projections (Figure 3A–3C), GFP+ sensory afferents in TauEWS-Pea3/+ Isl1Cre/+ embryos failed to invade the spinal cord and instead were found in an extreme lateral position at the dorsal root entry zone, a phenotype observed at least up to E18.5 (Figure 3A–3C and 3G–3I; data not shown). We next visualized the path of sensory afferent projections towards the dorsal root entry zone in TauEWS-Pea3/+ Isl1Cre/+ embryos by injecting fluorescently labeled dextran into an individual DRG (L3; n = 3; Figure 3M–3Q). Sensory afferents in E13.5 wild-type embryos bifurcated at their lateral spinal entry point, and projected rostrally and caudally over six or more segmental levels while gradually approaching the midline (Figure 3M). Sensory afferents in TauEWS-Pea3/+ Isl1Cre/+ embryos also bifurcated at the entry point, although approximately 5% of afferent fibers continued to grow towards the midline (Figure 3O and 3Q). While rostro-caudal projections were present in TauEWS-Pea3/+ Isl1Cre/+ embryos, afferent fibers failed to approach the midline at distal segments and continued to occupy an extreme lateral position (Figure 3O), consistent with the analysis of transverse sections.
Figure 3  Defects in the Establishment of Sensory Afferent Projections upon Precocious Expression of EWS-Pea3 in DRG Neurons
(A–C and G–I) Visualization of sensory afferent projections (green) into the spinal cord of wild-type (A–C) and TauEWS-Pea3/+ Isl1Cre/+ (G–I) embryos at E13.5 (A, C, G, and I) and E16.5 (B and H) by Cre-recombinase-mediated activation of mGFP expression from the Tau locus (A, B, G, and H) or by a Thy1spGFP transgene (C and I; [25]). Grey arrows indicate normal pattern of afferent projections into the spinal cord, whereas red arrows show aberrant accumulation of sensory afferents at the lateral edge of the spinal cord in TauEWS-Pea3/+ Isl1Cre/+ embryos.
(D–F and J–L) Analysis of sensory afferent projections (green) into the skin (D and J) or muscle (E and K; red, α-Bungarotoxin, BTX) of wild-type (D–F) and TauEWS-Pea3/+ Isl1Cre/+ (J–L) embryos at E16.5 by Cre-recombinase-mediated activation of mGFP (D, E, J, and K) expression from the Tau locus. (F and L) show Egr3 expression in intrafusal muscle fibers using in situ hybridization (consecutive sections to [E and K] are shown).
(M–Q) Analysis of bifurcation of sensory afferent projections towards the spinal cord in E13.5 wild-type (M) and TauEWS-Pea3/+ Isl1Cre/+ (O and Q) embryos after injection of fluorescently labeled dextran (green) into one DRG (lumbar level L3). Confocal scanning plane for (M and O) is schematically illustrated in (N). Inset in (O) is also shown at a deeper confocal scanning plane (P and Q) to visualize aberrant axonal projections.
Scale bar: (A and G), 60 μm; (B and H), 80 μm; (C and I), 100 μm; (D and J), 160 μm; (E, F, K, and L), 70 μm; (M, O, and Q), 240 μm. We next examined the establishment of peripheral projections upon precocious EWS-Pea3 expression in DRG neurons. While sensory axons in TauEWS-Pea3/+ Isl1Cre/+ embryos reached the skin and established major nerve trunks by E16.5, only rudimentary sensory axon branching was established within the skin (Figure 3D and 3J). In addition, there was a significant reduction in the number of muscle spindles in TauEWS-Pea3/+ Isl1Cre/+ embryos (approximately 25% of wild-type complement; n = 3) as assessed by innervation and expression of genes specific for intrafusal muscle fibers such as Egr3 (Figure 3E, 3F, 3K, and 3L; [26]). In summary, whereas isochronic expression of EWS-Pea3 promoted the establishment of proprioceptive afferent projections into the ventral spinal cord, precocious expression of the same ETS signaling factor in DRG neurons interfered with establishment of projections into the spinal cord as well as to peripheral targets.