The Nefl-Cre;NfascFlox mice were significantly smaller than their wild-type (+/+) littermates ( Figures 1D and 1E) and on average survived to P20. In order to test the specificity of Cre expression in neurons, the Nefl-Cre mice were crossed to the reporter MAPK inhibitor mouse strains TaumGFP/LacZ and Rosa26RLacZ(R26RLacZ) ( Hippenmeyer et al., 2005 and Soriano, 1999). It is important to note that two reporter strains were used, as the TaumGFP/LacZ line can only be expressed in neurons, while R26RLacZ is designed to be expressed

in any cell that expresses Cre. β-Galactosidase staining of Nefl-Cre;TaumGFP/LacZ tissue displayed positive activity of the Nefl promoter specifically in the brain, spinal cord, and dorsal root ganglia (DRG) neurons as early as P0, suggesting that Nefl-Cre is active prior to myelination ( Figure S1). At P11, both Nelf-Cre;R26RLacZ and Nefl-Cre;TaumGFP/LacZ mice showed robust neuron-specific expression in spinal cord and brain tissue, respectively, with no staining observed Apoptosis inhibitor within the white matter tracts ( Figure S1). Furthermore, we observed an increase in the expression of Nefl-Cre with time, suggesting that Nefl-Cre is temporally and dynamically regulated in neurons. To

further test the specificity of Nefl-Cre expression in neurons, sciatic nerves (SNs, not including the DRG) and spinal cords from P12 wild-type (+/+), Cnp-Cre;NfascFlox, and Nefl-Cre;NfascFlox mice were extracted Ketanserin and immunoblotted with antibodies against Cre recombinase (Cre) and pan-Neurofascin (NFct). Immunoblots of P12 SN lysates revealed strong expression of Cre in the glial-specific Cnp-Cre lysates (Cnp-Cre;NfascFlox), while both wild-type (+/+) and Nefl-Cre;NfascFlox lysates were negative for Cre expression ( Figure 1F). These results are consistent with previous reports ( Schweizer et al., 2002) and indicate that SCs do not express Nefl-Cre. Furthermore, spinal cord lysates from the same mice showed a significant reduction in neuronal

NF186 expression in Nefl-Cre;NfascFlox mice compared to wild-type (+/+), while glial NF155 expression remained unchanged ( Figure 1F). These results further confirm the specificity of Nefl-Cre expression in neurons alone. Next, immunoblot analysis of spinal cord lysates from P3, P6, P12, and P19 wild-type (+/+) and Nefl-Cre;NfascFlox age-matched littermates was carried out. A significant loss of NF186 was observed at P3 in Nefl-Cre;NfascFlox spinal cords and was sustained through P19, while NF155 expression increased with time in the Nefl-Cre;NfascFlox as in the wild-type (+/+) spinal cord lysates ( Figure 1G). Cre expression was present as early as P3 and persisted through P19 in Nefl-Cre;NfascFlox spinal cords.