In vivo and in vitro development of sensory neurons from Wnt1-cre conditional Shp2 mutant mice. Differentiation of sensory neurons in lumbar dorsal root ganglia was analyzed by immunohistochemistry using antibodies directed against (A and B) TrkA and Isl1/2, (C and D) TrkC and Brn3a in control and Wnt1-cre Shp2^fl/fl mice. (E) Ratios of TrkA+/Isl1/2+ and TrkC+/Isl1/2+ neurons at E11.5; the absolute number of Isl1/2+ neurons were similar in lumbar dorsal root ganglia at this stage (2537 ± 297 and 2101 ± 327 Isl1/2+ cells in control and Shp2 mutant ganglia, respectively; n = 4 and P = 0.074). (F and G) Cutaneous axonal projections of control and Wnt1-cre Shp2^fl/fl mice at E12.5, as revealed by whole mount immunohistochemistry using anti-L1 antibodies. (H) Quantification of the branching of cutanous axonal projections of control and Wnt1-cre Shp2^fl/fl mice. (I and J) Apoptosis of cells in lumbar dorsal root ganglia at E12.5 in control and Wnt1-cre Shp2^fl/fl mice, as assessed by TUNEL staining. (K and L) Appearance of lumbar dorsal root ganglia at E13.5 in control and Wnt1-cre Shp2^fl/fl mice, as assessed by immunohistochemistry using antibodies directed against Isl1/2. (M) Number of cells in dorsal root ganglia of control and Wnt1-cre Shp2^fl/fl mice at E11.5 and E13.5. (N and O) Dorsal root ganglia from E11.5 control and Wnt1-cre Shp2^fl/fl mice were cultured in the presence of Nrg1, NGF, and NT3 for 2 days; depicted are cultures stained with antibodies directed against NF160 and BFABP. (P and Q) Cultured dorsal root ganglia from control and Wnt1-cre Shp2^fl/fl mice; the ganglia were cultured for 2 days on a layer of Schwann cells obtained from wild-type mice in the presence of Nrg1, NGF, and NT3. (R) Quantification of axonal outgrowth observed in dorsal root ganglia cultures. [Scale bar, (A–D, I–L) 50 μm; (F and G) 150 μm; (N–Q) 200 μm.] *, P < 0.05; ***, P < 0.001.

Nrg1-evoked cellular and biochemical responses in Schwann cells treated with pharmacological inhibitors of Src, Mek, and Fak. (A and B) Quantification of Nrg1-induced migration and proliferation of control Schwann cells and Schwann cells treated with the Src inhibitor PP2, the Mek inhibitor U0126, and the Fak inhibitor TAE226. (C) Phosphorylation of Fak and Erk1/2 in the presence or absence of the Src inhibitor PP2. **, P < 0.01.

Severe deficits in Schwann cell development in Wnt1-cre conditional Shp2 mutant mice. Analysis of Schwann cells associated with peripheral nerves in control and Wnt1-cre Shp2^fl/fl mice at the indicated developmental stages using (A and B) immunohistochemistry for BFABP (red) and neurofilament (NF, green), (C and D) in situ hybridization with a Sox10-specific probe, and (E and F) histological sections stained with hematoxylin and eosin. [Scale bars, (A and B) 150 μm; (C–F) 50 μm.]

Nrg1-evoked cellular and biochemical responses in Shp2 mutant Schwann cells. Southern blot (A) and western blot (B) analyses after HTN-cre induced recombination in cultured Shp2^fl/fl Schwann cells. (C and D) Quantification of the Nrg1-induced proliferation and migration. Shown are the percentage of Schwann cells that incorporated BrdU or the percentage of Schwann cells that migrated into the scratched area, using control and Shp2^Δ/Δ Schwann cells (HTN-cre-treated Shp2^fl/fl Schwann cells) in the presence and absence of Nrg1. (E–F′) Migration of Schwann cells into a scratched area. (G, H, and I) Nrg1-induced phosphorylation of Erk1/2, Akt, Shc, Shp2, Src, and Fak in control and Shp2^Δ/Δ Schwann cells analyzed by western blot analysis. Depletion of Shp2 after HTN-cre-induced mutation was assessed in B and H; α-tubulin served as a loading control. [Scale bar, (E–F′) 50 μm.] **, P < 0.01.

Hypomyelination of peripheral nerves in Krox20-cre Shp2 mutant mice. (A and B) Electron microscopic analysis of control and Krox20-cre Shp2^fl/fl mice show a marked hypomyelination in the adult. (C) Quantification of myelin thickness in adult control, Krox20-cre Shp2^fl/fl, and Krox20-cre ErbB2^fl/fl mice. Displayed is a plot of the myelin thickness versus axon diameter. In each animal analyzed, 100 axons were counted. (D) Comparison of genes whose expression is deregulated in peripheral nerves of Krox20-cre Shp2^fl/fl and Krox20-cre ErbB2^fl/fl mutant mice at P8. The percentages of coregulated genes were determined using false discovery rates of 10⁻³ or 10⁻⁴ as cut-offs (see SI Methods). (E) Phosphorylation of Erk1/2, Akt, and Fak in peripheral nerves of control, Krox20-cre Shp2^fl/fl, and Krox20-cre ErbB2^fl/fl mutant mice at P8. [Scale bar, (A and B) 1 μm.]