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Fig. S1. Specificity of type III Nrg1 antibody. DRG from E17.5 WT (A) or type III Nrg1−/− (B) embryos were labeled for type III Nrg1 using an antibody generated against its cysteine-rich domain (Yang et al., 1998). Sensory neurons were labeled with anti-type III Nrg1 antibody in the WT but not in type III Nrg1 null embryos. Scale bar: 50 µm.
Fig. S2. Type III Nrg1 regulates neuron survival. (A) Percentages of TrkA+, TrkB+ and parvalbumin+ neurons per DRG in sections of E18.5 WT or type III Nrg1−/− DRG. Here, the data are in pie chart format to illustrate the relative proportions of each neuron type (see also Fig. 2 and Table S2 for absolute numbers). The sum of TrkA+, TrkB+ and parvalbumin+ neurons per DRG in each genotype was set to 100%, and the number of neurons for each of these markers was divided by the sum. The total area of the pie chart for each genotype is proportional to the total number of TrkA+, TrkB+ and parvalbumin+ neurons per DRG, which is stated below each graph. Type III Nrg1 deficiency resulted in an increased percentage of parvalbumin+ neurons and decreased percentages of TrkA+ and TrkB+ neurons per DRG. Mean ± s.e.m. (Ba-d) For assessment of apoptotic cell death, sensory neurons were maintained in serum-free medium with Ngf (25 ng/ml) for 24 hours. Neurons were then incubated in serum-free medium supplemented with B4-ECD (5 µg/ml) or Ngf (50 ng/ml) for 16 hours. B4-ECD treatment (16 hours) increased the survival of Ngf-deprived sensory neurons from WT (b) but not type III Nrg1−/− (d) embryos. Cell death was assessed by immunostaining for cl-Casp-3 (green), and neurons were identified by NeuN (Rbfox3) staining (red). Scale bar: 20 µm. (C) Quantification of cl-Casp-3+ neurons. Ngf treatment rescued WT and type III Nrg1 null neurons from cell death, whereas B4-ECD treatment rescued only WT neurons. Mean ± s.e.m. *, P<0.02; **, P<0.0002 (ANOVA with post-hoc Fisher�s PLSD test).
Fig. S3. Normal bifurcation of central sensory projections in type III Nrg1−/− embryos. Lumbar level spinal cords with intact DRG were removed from E15.5 embryos and fixed in 4% PFA overnight at 4°C. DRG were injected with DiI crystals (Molecular Probes) and incubated in 4% PFA for 5 days at 37°C. Spinal cords were analyzed as whole mounts, and labeling was captured with confocal microscopy. Bifurcation of sensory afferents in type III Nrg1−/− spinal cord (B) closely resembled that of WT (A) at E14.5. Injected DRG are outlined. Schematic representation of confocal scanning plane (C). Scale bar: 200 µm.
Fig. S4. Type III Nrg1 is required for TrkA+ axon pathfinding in the dorsal horn. Summary plots of individual TrkA+ axon termination positions (red dot) within the spinal cord of WT or type III Nrg1−/− embryos at E14.5 (green), E16.5 (yellow) and E18.5 (red). Quantification of axon termination positions along the dorsoventral axis for axons projecting into medial (gray) or lateral (black) regions of the spinal cord as a percentage of total axon termination points (WT, n=16 embryos; KO, n=17 embryos).
Fig. S5. Innervation of cutaneous targets is not sustained in type III Nrg1−/− embryos during late embryogenesis. (A-F) Transverse sections of hindlimb epithelium from WT (A-C) or type III Nrg1−/− (D-F) embryos at E14.5, E16.5 or E18.5 were labeled for peripherin. At E14.5, peripherin+ axons innervated the epidermis in WT (A) and mutant (D) embryos, but axons appeared thinner and disorganized in mutants. By E16.5 (E) and E18.5 (F), peripherin+ axon innervation in mutant embryos was greatly reduced compared with WT. Scale bar: 50 µm.
Fig. S6. Substance P expression does not require type III Nrg1. (A-D) Transverse sections of hindlimb epithelium at E17.5 (A,B) or lumbar DRG at E18.5 (C,D) from Bax−/−; type III Nrg1+/+ or Bax−/−; type III Nrg1−/− embryos were labeled for substance P. Substance P was detected in the epithelium (B) and DRG cell bodies (D) in the double-null mutant embryos. Arrows indicate substance P+ axons. Scale bars: 50 µm.
Fig. S7. Type III Nrg1 regulates plexin A4 expression. (Aa-f) DRG explants from E14.5 WT embryos were co-immunolabeled for type III Nrg1 and Nrp1 (a-c) or plexin A4 (d-f). Type III Nrg1 was co-expressed with Nrp1 (c) and plexin A4 (f) along sensory axons. Scale bar: 50 µm. (B) Sensory axons were isolated from cell bodies using Transwell membranes, and protein extracts from lower or upper Transwell membranes were assessed by immunoblotting to detect NeuN or peripherin. Protein extracts from sensory axons alone (lower membrane) contained peripherin but not NeuN, whereas extracts from upper membranes contained both peripherin and NeuN, indicating the presence of both sensory axons and cell bodies. (C) Immunostaining for NF160 (Nefm) on the lower membrane showed axons that crossed the Transwell membrane. Scale bar: 100 µm. (D) Type III Nrg1 affected the protein levels of the Sema3A co-receptor plexin A4. DRG explants from WT, type III Nrg1+/− or type III Nrg1−/− embryos were cultured on Transwell membranes, and protein extracts from the upper (total cell) and lower (axons only) membranes were collected and analyzed by immunoblotting. Plexin A4 levels were quantified and normalized to peripherin levels. WT and type III Nrg1+/− (HET) ratios were pooled and set to 1, and type III Nrg1−/− (KO) values are relative to this. Data points are presented in a scatter plot, with bar indicating the mean (n≥4 pups per genotype for littermate comparisons). *, P<0.0005 (Student�s t-test). (E) Type III Nrg1 expression did not affect TrkA protein levels. Total cell lysates from WT or type III Nrg1−/− DRG explants were assessed by immunoblotting to detect TrkA. Peripherin was used as a lysate loading control.