Figure 5.
Point-mutated DINEE613V fails to rescue the DINE KO phenotype. A, Constructs of Hb9:DINEE613V (TgE613V) transgenic mice. cDNA encoding DINEE613V was inserted downstream of an ∼9 kb Hb9 promoter, followed by IRES, EGFP, and polyA to express DINE and EGFP simultaneously in embryonic spinal motor neurons. In the DINEE613V construct, Glu613 (E) was converted to Val (V). B, Coronal section of the spinal cord at E17.5 of TgE613V mouse was immunostained with anti-GFP (green) and anti-ChAT (red) antibodies. Most of ChAT-positive motor neurons expressed exogenous GFP specifically in spinal motor neurons. C, Coronal sections of the spinal cord at E17.5 of KO;TgWT and KO;TgE613V mice were immunostained with anti-DINE antibody (green). D, Whole-mount immunohistochemistry reveals the phrenic nerve arbor in the diaphragm (top) and the thoracodorsal nerve arbor in LD (bottom) of WT, KO, KO;TgWT, and KO;TgE613V mice at E17.5 using anti-peripherin (green) antibody and BTX (red). E, F, Total length of phrenic nerve (E) or thoracodorsal nerve (F) was determined as the sum of each branch length distal to the branching point (* in D) at E17.5. **p < 0.01. *p < 0.1. cc, Central canal. Scale bars: B, C, 200 μm; D, 500 μm. n.s., Not significant. Error bars indicate SD.