Figure 1. Generation of DINEWT and DINEmut transgenic mice. A, Phenotype observed in spinal cord and diaphragm of DINE KO mice. Top column, DINE immunoreactivity observed in embryonic spinal cord (E17.5) in WT and DINE-deficient (KO) mice. Bottom column, Whole-mount preparation of diaphragm of WT and KO embryos at E17.5, indicating the phrenic nerve arbor. Anti-peripherin antibody (green) and BTX (red) were used to identify nerves and synaptic structures of NMJ, respectively. No DINE-positive motor neurons are observed in the spinal cord of KO mice, and the phrenic nerve arbor is strikingly poor in the diaphragm of KO mice. Asterisks indicate branching point. B, Constructs of Hb9:DINEWT (TgWT) and Hb9:DINEmut (Tgmut) transgenic mice. Either cDNA encoding DINEWT or DINEmut 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 DINEmut construct, 5 amino acids (HELTH) were deleted and C-terminal Glu672 (E) was converted to Val (V). C, Coronal sections of the spinal cord at E17.5 of TgWT and Tgmut mice. The sections were immunostained with anti-GFP (green) and anti-ChAT (red) antibodies. Both transgenic mice express GFP specifically in spinal motor neurons, and GFP intensity appears to be similar between TgWT and Tgmut mice. D, The ratio of GFP-positive cells in ChAT-positive motor neurons is similar between TgWT and Tgmut mice. Almost all motor neurons in the cervical spinal cord at E17.5 express the transgene in both Tg mice. No statistically significant difference was seen. E, Western blotting analysis of membrane (M) and cytosol (C) fractions using ventral spinal cord lysate from WT, KO, TgWT, and Tgmut embryos at E13.5. N-Cadherin (N-Cadh) and MEK1/2 were used as positive controls for membrane and cytosol protein, respectively. F, Sucrose gradient fractionation using ventral spinal cord lysate from TgWT, and Tgmut embryos at E13.5. Calreticulin and N-Cadh were used as ER and plasma membrane markers, respectively. G, Whole-mount immunohistochemistry of diaphragm was performed using E17.5 TgWT and Tgmut mice. Both TgWT and Tgmut mice exhibit similar arborization patterns of the phrenic nerve compared with WT mice. H, Total length of the phrenic nerve, which is the sum of each branch length distal to the branching point (* in G), was measured using diaphragms from E17.5 WT, KO, TgWT, and Tgmut mice. There are no significant differences between WT, TgWT, and Tgmut mice. **p < 0.01. cc, Central canal. Scale bars: A, C, 200 μm; G, 500 μm. n.s., Not significant. Error bars indicate SD.