Figure 3. Runx1 functions downstream of NGF to mediate expression of the majority of nonpeptidergic nociceptor-specific genes, whereas it controls Ret expression at least in part by enhancing NGF signaling.

(A–L) In situ hybridization analysis of expression of Mrgprd (A–C), Gfra2 (D–F), Ptprt (G–I) and Ret (J–L) in DRGs of P2 control animals that received BSA injections, Runx1CKO animals that received BSA injections or Runx1 CKO animals that received NGF injections. Note that exogenous NGF administration fails to activate expression of nonpeptidergic-specific genes in Runx1 CKO animals, with the notable exception of Ret, suggesting that Runx1 is a downstream mediator of NGF signaling that is required for expression of the majority of nonpeptidergic-specific genes. The ability of exogenous NGF to upregulate Ret expression in Runx1 CKO animals is most consistent with an indirect role for Runx1 in regulating Ret expression, through enabling NGF signaling. Shown are results representative of at least three independent injection experiments. See also Figure 3—figure supplements 1, 2. (M–P) Real-time PCR analysis of expression of Mrgprd (M), Gfra2 (N), Ptprt (O) and Ret (P) in dissociated DRG neurons from P0 control and Runx1 CKO animals cultured in the presence or absence of NGF. Note that, with the exception of Ret, NGF-dependent expression of these nonpeptidergic-specific genes is completely abolished in the absence of Runx1, further supporting Runx1 as a downstream mediator of NGF in regulating expression of most nonpeptidergic-specific genes. Statistical analyses were done using two-way ANOVA with a Bonferroni post-test, N = 5 for M and P, N = 7 for the rest. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns non-significant. Runx1^f/f mice were used as control animals for analysis of Runx1 CKO mutants. Scale bar, 50 μm.

DOI:http://dx.doi.org/10.7554/eLife.10874.006

Figure 3—figure supplement 1. Runx1 potentiates TrkA activity without regulating TrkA expression.

(A–D) Double staining of pTrk-SHC and Neurofilament heavy chain (NFH) (A and B) or pTrk-PLCγ and NFH (C and D) in control and Runx1 CKO DRGs at P0 shows greatly diminished pTrk immunoreactivity in NFH-negative neurons in Runx1 CKO DRGs compared to controls, suggesting Runx1-dependence of NGF signaling at this time point. NFH was used to exclude myelinated TrkB, TrkC-expressing DRG neurons from analysis. (E and F) Quantification of the NGF signaling deficit based on average fluorescence intensity of pTrk-SHC or pTrk-PLCγ immunoreactivity per cell within the neuronal population that is negative for NFH. An unpaired t test was performed on data from three independent pairs of control and mutant animals, **p ≤ 0.01, ***p ≤ 0.001. (G and H) TrkA immunostaining in control and Runx1 CKO DRGs at P0 shows comparable expression in both genotypes. Shown are results representative of three independent animals per genotype. Scale bar, 50 μm.

DOI:http://dx.doi.org/10.7554/eLife.10874.007

Figure 3—figure supplement 2. Runx1 controls expression of the majority of nonpeptidergic-specific genes independent of its stimulatory effect on NGF signaling.

(A–D) Real-time PCR analysis of expression of Mrgprd (A), Gfra2 (B), Ptprt (C) and Ret (D) in DRGs of P2 control animals that received BSA injections, Runx1CKO animals that received BSA injections or Runx1 CKO animals that received NGF injections. A one-way ANOVA with a Tukey's multiple comparison test was performed on data from three independent experiments, *p ≤ 0.05, ns non-significant.

DOI:http://dx.doi.org/10.7554/eLife.10874.008