Figure 7. NGF promotes Cbfb expression through the ERK/MAPK signaling pathway.

(A–D) Double staining of Flag (green) and βIII-Tubulin (blue) in DMSO or U0126-treated dissociated DRG neurons from P0 Cbfb^Flag/+ animals that were cultured without or with NGF. U0126 is a selective inhibitor of MEK1/2, the direct activators of ERK1/2. Note that CBFβ protein expression, as defined by Flag immunoreactivity is greatly diminished in U0126-treated neurons as compared to vehicle-treated neurons, all grown in the presence of NGF. (E) Quantification of the effect of U0126 treatment on CBFβ protein levels based on experiments as described in (A–D). CBFβ protein abundance was quantified as the average fluorescence intensity of Flag immunoreactivity per cell. Statistical analysis was done using a two-way ANOVA with a Bonferroni post-test, based on data from four independent experiments. ***p ≤ 0.001, ns non-significant. See also Figure 7—figure supplement 1. (F and G) In situ hybridization analysis of Cbfb expression in control and quadruple DRGs at P0 reveals a severe deficit in Cbfb mRNA expression in DRGs when MAPK signaling is disrupted in the nervous system. A similar phenotype of varied severity was observed in 4 out of 5 mutant animals. (H–J) In situ hybridization analysis of Cbfb expression in Ntrk1 ^+/-; Bax^-/-, Ntrk1^-/-; Bax^-/- and Ntrk1^-/-; Bax^-/-; Nes-Cre; V600E DRGs at E18.5 shows that constitutive activation of MAPK signaling leads to a dramatic increase in Cbfb expression in TrkA-deficient animals. Shown are representative images from two independent experiments. (K) Real-time PCR analysis of Cbfb expression in the same set of loss-of-function and gain-of-function mouse models as described in (F and G) and (H–J) at P0 further demonstrates the necessity and sufficiency of MAPK signaling for NGF-dependent Cbfb expression in vivo. Shown are averages from two independent experiments after normalization to littermate control. Scale bar, 50 μm.

DOI:http://dx.doi.org/10.7554/eLife.10874.015

Figure 7—figure supplement 1. In vitro evidence for the necessity of MAPK signaling for NGF-dependent CBFβ expression.

(A) Immunoblot analysis of expression of Cbfb in DMSO or U0126-treated dissociated DRG neurons from P0 Cbfb^{Flag/ +} animals that were cultured in the presence or absence of NGF. Histone 3 serves as a protein loading control. (B and C) Quantification of the effect of U0126 on NGF dependence of Flag-CBFβ or CBFβ expression based on (A). Inactivation of MAPK signaling by U0126 attenuates NGF-dependent CBFβ expression. The levels of Flag-CBFβ or CBFβ protein were determined by densitometry, and normalized to that of Histone 3. The relative protein abundance of Flag-CBFβ or CBFβ after normalization to the level in DMSO-treated cultures that were grown without NGF was subject to a two-way ANOVA test with a Bonferroni post-test, N = 3. *p ≤ 0.05, **p ≤ 0.01, ns non-significant.

DOI:http://dx.doi.org/10.7554/eLife.10874.016