Precocious EWS-Pea3 Expression Promotes Neurotrophin-Independent Survival and Neurite Outgrowth
To begin to address the cellular and molecular mechanisms involved in the distinct biological actions of EWS-Pea3 at different developmental stages, we first turned to in vitro culture experiments. These experiments permit assessment of whether precocious ETS transcription factor signaling influences neuronal survival and in vitro neurite outgrowth of DRG neurons, two parameters prominently influenced by target-derived neurotrophic factors and their receptors.
We cultured E13.5 whole DRG explants from wild-type and TauEWS-Pea3/+ Isl1Cre/+ embryos in the presence of NGF or NT-3 or in the absence of neurotrophins and analyzed neuronal survival and neurite outgrowth on matrigel substrate after 48 h in vitro. Without neurotrophic support, very few wild-type DRG neurons survived (Figure 4A). In contrast, culturing wild-type DRG with neurotrophic factors led to neuronal survival and neurite outgrowth. Addition of NGF, which supports survival of cutaneous afferents, resulted in straight and unbranched neurite outgrowth (Figure 4B), while cultures grown in the presence of NT-3, which supports survival of proprioceptive afferents, resulted in a highly branched neurite outgrowth pattern after 48 h in vitro (Figure 4C). Surprisingly, DRG neurons isolated from TauEWS-Pea3/+ Isl1Cre/+ embryos and cultured without neurotrophic support survived after 48 h in vitro and had established long and highly branched neurites (Figure 4D). Neither the pattern of neurite outgrowth nor neuronal survival changed significantly after application of either NGF or NT-3 (Figure 4E and 4F).
Figure 4  Neurotrophin-Independent Neurite Outgrowth In Vitro of DRG Neurons Expressing EWS-Pea3 Precociously
E13.5 lumbar DRG from wild-type (A, B, and C), TauEWS-Pea3/+ Isl1Cre/+ (D, E, and F), or Bax−/− (G, H, and I) embryos cultured for 48 h without neurotrophic support (A, D, and G) or in the presence of NGF (B, E, and H) or NT-3 (C, F, and I) were stained for expression of neurofilament to visualize axonal extensions.
Scale bar: 130 μm. To directly compare neurotrophin dependence of DRG neurons expressing EWS-Pea3 from the Tau locus at a precocious versus isochronic time of onset, we generated a strain of mice in which Cre recombinase is expressed from the PV locus (Figure S4). The expression of GFP in TaumGFP/+ PVCre/+ was restricted to PV+ proprioceptive DRG neurons and mirrored the onset of expression of PV at approximately E14 (Figure S4; data not shown). We next cultured E14.5 whole DRG explants from TauEWS-Pea3/+ PVCre/+ and TaumGFP/+ PVCre/+ mice for 48 h in vitro in the presence or absence of NT-3 (Figure 5). We found that DRG neurons from both genotypes survived and extended neurites only in the presence of NT-3, whereas they died in the absence of NT-3 (Figure 5). Together, these findings suggest that only precocious but not isochronic ETS signaling in DRG neurons is capable of uncoupling survival and neurite outgrowth from a requirement for neurotrophin signaling normally observed in wild-type DRG.
Figure 5  DRG Neurons Expressing EWS-Pea3 Isochronically Depend on Neurotrophins for Survival
E14.5 lumbar DRG from TaumGFP/+ PVCre/+ (A and B) and TauEWS-Pea3/+ PVCre/+ (C and D) embryos cultured for 48 h without neurotrophic support (A and C) or in the presence of NT-3 (B and D) were stained for expression of neurofilament (red) and LacZ (green) to visualize axonal extensions and survival of PV-expressing proprioceptive afferents.
Scale bar: 150 μm. To determine whether neuronal survival of DRG neurons from TauEWS-Pea3/+ Isl1Cre/+ embryos in the absence of neurotrophic support is sufficient to explain the observed neuronal outgrowth, we analyzed DRG isolated from mice mutant in the proapoptotic gene Bax [27]. Consistent with previous results, Bax−/− DRG neurons survived without neurotrophic support [28]. In contrast, neurite outgrowth of Bax−/− DRG neurons was significantly less (see Figure 4G) than that of either DRG from TauEWS-Pea3/+ Isl1Cre/+ embryos cultured in the absence of neurotrophic support (see Figure 4D) or Bax−/− DRG neurons cultured in the presence of neurotrophic support (see Figure 4H and 4I). These findings suggest that in addition to mediating neurotrophin-independent neuronal survival, expression of EWS-Pea3 in early post-mitotic neurons also promotes neurite outgrowth in a neurotrophin-independent manner.
To begin to assess at which step of the neurotrophin signaling cascade DRG neurons from TauEWS-Pea3/+ Isl1Cre/+ embryos become unresponsive to the addition of neurotrophins, we assayed the expression of neurotrophin receptors in TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure 6). Whereas expression of the neurotrophin receptors TrkA, TrkB, and TrkC marks afferents of distinct sensory modalities in DRG of wild-type embryos (Figure 6A–6C) [4,29], TauEWS-Pea3/+ Isl1Cre/+ embryos showed complete absence of expression of TrkA, TrkB, and TrkC in DRG neurons at E16.5 (Figure 6G–6I). This absence of Trk receptor expression in DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos provides a likely explanation for the lack of responsiveness of these neurons to the addition of neurotrophic factors.
Figure 6  Loss of Trk Receptor Expression and Increased Survival in DRG Neurons upon Precocious ETS Signaling
(A–C and G–I) In situ hybridization analysis of TrkA (A and G), TrkB (B and H), and TrkC (C and I) expression in E16.5 lumbar DRG of wild-type (A–C) and TauEWS-Pea3/+ Isl1Cre/+ (G–I) embryos.
(D–F and J–L) Analysis of lumbar DRG of wild-type (D), TaumGFP/+ IslCre/+ (E and F), and TauEWS-Pea3/+ Isl1Cre/+ (J, K, and L) embryos for (1) neuronal cell death at E13.5 by TUNEL (green; D and J), (2) cell survival and proliferation at E16.5 by LacZ (blue) wholemount staining (E and K; lumbar levels L1 and L2 are shown), and (3) BrdU (green)/LacZ (red) double labeling (F and L).
(M and N) Quantitative analysis (n ≥ 3 independent experiments) of the mean number of apoptotic events relative to wild-type levels is shown in (M) and neuronal survival in (N) as percent of wild-type of DRG at lumbar levels L1 to L5 as quantified on serial sections.
(O) Western blot analysis of protein extracts isolated from lumbar DRG of E16.5 wild-type (wt) and TauEWS-Pea3/+ Isl1Cre/+ (mut) embryos using the following antibodies: Akt, p-Akt (Ser473), CREB, p-CREB (Ser133), Bax, Bcl2, and Bcl-xl.
(P) Quantitative analysis of protein levels relative to wild-type in percent is shown on the right (n = 3 independent experiments).
Scale bar: (A–C and G–I), 35 μm; (D and J), 40 μm; (E and K), 200 μm; (F and L), 50 μm. We next assayed whether the complete absence of Trk receptor expression in TauEWS-Pea3/+ Isl1Cre/+ embryos had an influence on naturally occurring cell death in vivo using TUNEL on DRG sections. Surprisingly, we found that apoptosis was decreased by approximately 50% (n = 3 embryos, average of >50 sections) in DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos in comparison to wild-type (Figure 6D, 6J, and 6M). Quantitative analysis of the number of neurons in lumbar DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos revealed a significant increase to approximately 170% of wild-type levels (Figure 6E, 6K, and 6N). Moreover, BrdU pulse-chase experiments ruled out the possibility that DRG neurons in TauEWS-Pea3/+ Isl1Cre/+ embryos reenter the cell cycle (no BrdU+/LacZ+ cells, n = 3 embryos, analysis of >50 sections each; Figure 6F and 6L). Together with the in vitro culture experiments, these findings suggest that DRG neurons from TauEWS-Pea3/+ Isl1Cre/+ embryos remain post-mitotic but fail to become sensitive to naturally occurring cell death, and survive in the absence of Trk receptors and neurotrophic support.
We next analyzed whether changes in the expression of proteins known to be involved in the regulation of neuronal survival or cell death could be detected in DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos. We found no significant quantitative changes in the level of Akt/p-Akt or CREB/p-CREB in DRG (Figure 6O and 6P) both of which have been shown to be key regulators of neuronal survival [29]. Moreover, the level of the proapoptotic Bcl2 family member Bax was not significantly reduced (Figure 6O and 6P). In contrast, the expression level of the anti-apoptotic Bcl2 family members Bcl-xl and Bcl2 was significantly increased when compared to wild-type levels (Bcl2, 157%; Bcl-xl, 259%; average of n = 3 independent experiments; Figure 6O and 6P), providing a potential molecular explanation for the enhanced neuronal survival of DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos in the absence of Trk receptor expression [30].