Article Figures & Data
Figures
- Figure 1.
GRPs enhance axonal growth in DRGs in vitro. A–C, Representative montage images are shown for control dissociated DRG culture (A), dissociated DRGs cocultured with rat GRPs (B), and dissociated DRGs cultures exposed to conditioned medium from alkaline phosphatase-expressing rat GRPs (C). βIII-tubulin (red) immunofluorescence highlights the neurons; GRPs are visualized by immunostaining for alkaline phosphatase (green). D shows quantification for the average length of the longest axon per neuron ± SEM (n ≥ 30 neurons in three separate experiments; **p ≤ 0.01 by Student’s t test). Scale bar, 250 μm.
- Figure 2.
Coculture with neural and glial progenitor cells increases axonal outgrowth from adult DRG neurons. A–C, Representative montage images of control-dissociated DRG cultures (A), dissociated DRGs cocultured with rat GRPs/NRPs (B), and dissociated DRG cultures exposed to conditioned medium from parallel rat GRP/NRP cultures (C) are shown. Immunofluorescence for βIII-tubulin (red) and nestin (green) highlight DRG neurons and GRP/NRP nuclei, respectively. D, Quantitation of the average lengths of the longest axon per neuron (±SEM) for the above conditions is shown. Coculture with GRPs/NRPs significantly increases in axon length compared with the standard DRG culture; exposure to conditioned medium from GRP/NRP cultures showed a further increase in axon length (n ≥ 30 neurons in three separate experiments; *p ≤ 0.05 and ***p ≤ 0.001 by Student’s t test). Scale bar, 250 μm. E, Quantitation of axon growth parameters for 7 d injury-conditioned DRG neurons cultured on coverslips laid over a bed of GRP/NRP cells (coculture) or control conditions is shown (n ≥ 200 neurons analyzed in three separate experiments; p values represent ANOVA with Tukey post hoc analyses).
- Figure 3.
Approach for DRG/progenitor cell coculture to isolate axonal processes. A, Schematic of modified Boyden chamber used for culture system for isolation of axons from DRG neurons is shown. For this, DRG explants were plated onto the upper surface of a porous PET membrane, as previously used for dissociated DRGs (Zheng et al., 2001), and GRPs/NRPs were cultured on the surface of the plate well. B–D, Representative confocal projection images of upper membrane surface with DRGs (B), corresponding image stacks for lower membrane surface showing a dense array of axons (C), and GRPs/NRPs along the plate surface (D) are shown. DRGs were stained for βIII-tubulin (green) and DAPI (blue). The GRPs were visualized with DiO stain (cyan). The insets in B and C show mCherry signals (red) of AAV5-mChMYR3'amph-transduced DRGs in cell bodies (B) extending into axons along the lower membrane surface (C). Scale bars: B, C, 100 µm; D, 50 µm. E, Representative RT-PCR from cell body and axonal isolates for DRGs cultured under control conditions or over a bed of GRPs/NRPs. The axonal isolates are depleted of cell body (MAP2 and c-Jun) and glial (GFAP) mRNAs, but contain the known axonal transcript β-actin.
- Figure 4.
Exposure to GRPs/NRPs alters the levels of growth-associated mRNA cell bodies and axons of adult DRG neurons. A, B, The modified coculture system from Figure 3 was used for analyses of RNA levels from the explanted ganglia (“cell body RNA”) and their extended axons (“axonal RNA”). Data from RT-ddPCR analyses with cell body and axonal RNA preparations for known axonal mRNAs are shown in A and B, respectively. Values are shown for the NRP/GRP coculture relative to control samples, as indicated, ±SD for biological replicates (N = 4; p ≤ 0.05 for indicated columns; &no positive droplets were detected for Reg3a amplification despite highest template input levels). C, Representative FISH/IF images of cell bodies and axon shafts are shown for indicated mRNAs for dissociated DRGs cultured under control or GRP/NRP-exposed conditions. For these studies, DRGs were cultured on coverslips over a bed of GRP/NRP cells. Image pairs for control and GRP/NRP conditions are exposure matched, and axonal mRNA signals are shown as a spectral intensity in the upper rows of axon image sequences and cell body, and lower axon panels show merged images for mRNA and NF. Scale bars: cell body panels, 25 µm; axon panels, 10 µm. D, Quantification of axonal FISH signal intensities from matched exposure images are shown for GRP/NRP coculture vs control cultures as the fold change ± SEM (n > 50 for axons and n > 25 for cell bodies in three culture preparations; p values are from ANOVA with Tukey post hoc analyses).
- Figure 5.
Alterations in axonal mRNA levels with GRP/NRP coculture is conferred by axonal mRNA UTRs. Aa–i, Representative fluorescent images for eGFP mRNA (red) and βIII-tubulin protein (green) in cell body and distal axon shaft of dissociated DRG cultures transfected with eGFPMYR plasmids with 3'UTRs of rat GAP43 (a, b), β-actin (c, d), CALR (e, f), and γ-actin mRNAs (g, h). i, Images for sense eGFP probe represent the 3'β-actin construct. Left-hand columns of axon and cell body images show control DRG cultures, and right-hand columns show DRGs cocultured with GRPs/NRPs. All image pairs are exposure matched (control vs GRP/NRP coculture axon and control vs GRP/NRP coculture cell body). As previously published, axonal GFP mRNA is not seen for the construct with 3'UTR of γ-actin, but axonal signals are seen for GAP43, calreticulin, and β-actin 3'UTR constructs (Willis et al., 2007; Vuppalanchi et al., 2010; Yoo et al., 2013). B, C, Quantitation of axonal and cell body GFP mRNA intensities across multiple transfection experiments for DRGs with or without GRPs/NRPs are shown as average signal intensities ± SEM (n ≥ 30 processes over three independent transfections and cultures; ***p ≤ 0.001 by Student’s t test). Scale bars, 20 µm.
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