ActRIIA and BMPRII are expressed by processes, growth cones and cell bodies, whereas ActRIIB is expressed primarily in growth cones. Therefore, the variety II receptors vital for BMP7 evoked chemotaxis, ActRIIA and BMPRII, are promi nently expressed by dI neurons. BMP7 mediated dI inductive specification is independent of PI3K signaling The observations described in the prior section prompted us to consider the roles of signaling pathways connected with type II BMP receptors in BMP7 evoked neuronal activities. We explored the possibility that a pathway mediated by PI3K could possibly elicit axon orientation independently of inductive specification. PI3K and LIMK1 are each connected with type II BMP receptors.
Furthermore, cell migration and chemotaxis of non neuronal cells in response to BMP2 and BMP7 are dependent inhibitor NVP-BEZ235 on PI3K, whereas LIMK seems to regulate price, but not path of, dI axon extension within the spinal cord. We therefore examined the role of PI3K activity in BMP evoked inductive specification and axon orientation in spinal explants, using the inhibitors of PI3K activity, LY294002 and wortmannin. As shown above, BMP7 and BMP6 stimu lated phosphorylation of Smad1 5 eight and induction of Lhx2 9 in explants. Incubation of explants with LY had no effect on BMP7 evoked pSmad1 5 eight or Lhx2 9 induction. Similarly, in explants, LY treatment had no effect on the inductive response to BMP7, co culture of BMP7 expressing COS 1 cells with explants induced ectopic Lhx2 9 expression. In the presence of LY, explants exposed to BMP7 showed a 5.
3 fold boost in Lhx2 9 expression that was not signifi cantly different from explants with no LY. With each other, these outcomes provide evidence that neither the phosphorylation of Smad1 5 8 nor the intra cellular events more hints underlying neural specification by BMP7 employ PI3K as a signaling intermediate. PI3K involvement in BMP7 mediated growth cone collapse and axon orientation We subsequent measured the effect of LY on BMP7 evoked axon orientation in the similar explants in which Lhx2 9 expression was monitored. In con trol explants co cultured with adjacent pellets of COS 1 cells expressing empty vector, axons extended using a straight D V trajectory with an angle of reorien tation of 0. 8 1.7. In explants adja cent to BMP7 expressing COS 1 cells, axons had been repelled, extending away in the BMP source with an angle of reorientation of 32. five 1. 9. LY considerably inhibited the potential of BMP7 to orient dI axons, in the presence of LY the angle of reor ientation in response to BMP7 was 11. 6 1.