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. 2016 Apr 27;36(17):4744–4757. doi: 10.1523/JNEUROSCI.3811-15.2016

Figure 2.

Figure 2.

Exogenous DINEWT expression rescues aberrant arborization of phrenic nerves observed in KO mice, whereas DINEmut expression fails to rescue the KO phenotype. A, Whole-mount immunohistochemistry reveals the phrenic nerve arbor in the diaphragm of WT, KO, KO;TgWT, and KO;Tgmut mice at E13.5 (top), E15.5 (middle), and E17.5 (bottom) using anti-peripherin (green) antibody and BTX (red). The phrenic nerve of KO;TgWT mice moderately recovers intramuscular arborization and forms a significant number of NMJs. However, the KO;Tgmut mice are lacking arborization throughout all examined embryonic stages and exhibit a similar pattern to the DINE KO mouse. B, Total length of phrenic nerve was determined as the sum of each branch length distal to the branching point (* in A) at E13.5, E15.5, and E17.5. Although recovery of the total length appears slightly worse in E13.5 and E15.5 of KO;TgWT mice, the nerve length is recovered in E17.5. Conversely, the KO;Tgmut mouse exhibits no recovery at all stages. t test compared with WT of equivalent embryonic day was performed. *p < 0.05. **p < 0.01. C, Sholl analysis of phrenic nerve arborization was performed using the diaphragm of E17.5 WT, KO, KO;TgWT, and KO;Tgmut mice. Nerve arbor complexity of KO;TgWT mice is as similar as that of WT mice, whereas it was significantly less in KO;Tgmut mice. **p < 0.01. D, Coronal sections of cervical spinal cord at E17.5 of WT, KO, KO;TgWT, and KO;Tgmut mice were immunostained by anti-DINE (green) and anti-Oct-6 (red) antibodies for detection of either endogenous or exogenous DINE and phrenic motor neurons, respectively. Expression of exogenous DINE was detected in spinal motor neurons in KO;TgWT and KO;Tgmut mice, but the expression was slightly weak in phrenic motor neurons labeled with Oct-6 (arrows) compared with endogenous DINE in WT mice. E, The number of ChAT-positive motor neurons in the cervical spinal cord was quantified, with no significant difference between WT, KO, KO;TgWT, and KO;Tgmut mice at E17.5. cc, Central canal. Scale bars: A, 500 μm; D, 200 μm. n.s., Not significant. Error bars indicate SD.