Figure 5

Cell autonomous regulation of MN survival by Met can be achieved through distinct signalling pathways. (A) In vitro assessment of survival of MNs derived from WT, met^2P/2P, met^2S/2S, and met^d/d E12.5 embryos in the presence of GDNF (100 pg/ml). Survival values are expressed as percentage of the survival in basal medium (defined as 100%). The ability of MNs to respond to GDNF is not altered in met^d/d, met^2P/2P, and met^2S/2S mutants. Therefore, in subsequent panels, values are normalised with the survival in basal medium (defined as 0%) and expressed as percentage of their response to GDNF (defined as 100%). (B,C) Survival of brachial + lumbar (B) or thoracic (C) MNs derived from WT, met^2P/2P, met^2S/2S, and met^d/d E12.5 embryos in the presence of HGF (2 ng/ml). Whereas met^d/d MNs do not show any survival response to HGF, met^2P/2P and met^2S/2S MNs respond as efficiently as WT MNs. (D) Signalling requirements downstream of Met^WT, Met^2P, and Met^2S for MN survival by HGF were explored using inhibitors of PI3K (LY294002: 1 μM), Mek (PD98059: 1 μM), and Src (PP2: 0.2 μM), respectively). Inhibitors were used at concentrations not toxic on the basal survival. All three inhibitors efficiently blocked the survival response of WT MNs to HGF. As expected, the survival response in met^2P/2P MNs was selectively blocked by the PI3K inhibitor. In contrast, the survival response in met^2S/2S MNs was abolished by inhibiting Src or Mek. For each genotype, two to four independent experiments were done in triplicate comparing MNs from mutant embryos and from their WT littermates. (WT: n=9; , met^2P/2P: n=3; met^2S/2S: n=4; met^d/d: n=2). Error bars indicate standard error of the mean. (E) Schematic representation of the signalling components involved in blocking apoptosis downstream of the wild-type Met^WT or the specificity switch mutants Met^2P and Met^2S receptors. The inhibitors used are indicated.