I-BMP4 antibody (E and F), or an NT-157 site anti-neurofilament antibody to mark axons (G), and visualized using a color reaction product (AEC). The proximal and distal ligations are marked in (E) by “p” and “d”, respectively. (F) 
Higher magnification 22948146 of the proximal region illustrated in (E). (H) A single section was double-stained with anti-BMP4 (red) and anti-neurofilament (green) antibodies to show colocalization of BMP4 immunoreactivity and axons. Red and green channels were merged using Adobe Photoshop software. The Calcitonin (salmon) site arrows and arrowheads point to Schwann cells and axons, respectively. Magnifications in A and E; B, C and G; D and H are the same. Scale bars = 200 mm (A and E) and 50 mm (B , F ). doi:10.1371/journal.pone.0058441.gRegulation of muscle-derived BMPMuscle-derived signals are essential for both developing and mature motor neurons. Although the target dependence only exists until early postnatal stage (P7 to P10) for the survival of developing motor neurons [27], mature motor neurons continue to receive some neurotrophic signals from adult muscle fibers. For instance, adult motor neurons receive muscle derived neurotrophin-4 (NT-4) for nerve growth and remodeling, although the expression of NT-4 in muscle fibers requires signals from motor neurons [28]. Equally, the disappearance of BMP4 immunoreactivity in muscle fibers following nerve transaction may suggest that BMP4 expression is regulated by motor neurons in a fashion similar to that of NT-4 (Fig. 2). In addition to agrin, we speculate that other unknown factors may be involved in this process. The neuronal regulation of BMP4 may reflect its function in the regulation of neuron survival, axon growth, and maintenance of synaptic connections as reported for NT-4 previously [28,29]. Although it is not clear how agrin regulates BMP4 expression in muscle cells, the activation of Lrp4/Musk receptor complex by agrin has been shown to stimulate JNK pathways which may further induce BMP4 expression [30,31]. The up-regulation of BMP4 in skeletal muscles after nerve ligation could reflect a similar physiological function for BMP4 to that discussed above. Indeed, it has been shown that enhancedFigure 5. Expression of BMP4 mRNA in the neuromuscular system following nerve ligation. Total RNA was isolated from sciatic nerve (n = 4), soleus muscle (n = 6) and lumbar spinal cord (n = 6) of control and nerve-ligated mice. Expression levels of BMP4 mRNA were measured by real-time PCR. Data are presented using GAPDH expression level as 100 . Values are mean 6 SEM (*, P,0.05, compared to normal mice using Student’s t test). doi:10.1371/journal.pone.0058441.gBMP4 and Motor NeuronFigure 6. BMP4 promotes embryonic motor neuron survival and protects NG108-15 neurons against glutamate (Glu)-induced excitotoxicity (Extt). (A) A dose-response curve for the survival effect of BMP4 on embryonic motor neurons. The numbers of surviving motor neurons were determined by counting 
islet-1 positive neurons under a microscope. Data are presented using cell counts in the vehicle group as 100 . (B ) Differentiated NG108-15 cells were treated with glutamate and/or BMP4 for 16 hours, followed by an 8-hour recovery period in conditioned medium with or without BMP4. Three representative photos show the morphology of differentiated NG108-15 neurons in control (C), glutamate-treated (D) and glutamate plus BMP4-treated (E) conditions. Scale bars = 100 mm. The numbers of surviving neurons were counted under a microscope by.I-BMP4 antibody (E and F), or an anti-neurofilament antibody to mark axons (G), and visualized using a color reaction product (AEC). The proximal and distal ligations are marked in (E) by “p” and “d”, respectively. (F) Higher magnification 22948146 of the proximal region illustrated in (E). (H) A single section was double-stained with anti-BMP4 (red) and anti-neurofilament (green) antibodies to show colocalization of BMP4 immunoreactivity and axons. Red and green channels were merged using Adobe Photoshop software. The arrows and arrowheads point to Schwann cells and axons, respectively. Magnifications in A and E; B, C and G; D and H are the same. Scale bars = 200 mm (A and E) and 50 mm (B , F ). doi:10.1371/journal.pone.0058441.gRegulation of muscle-derived BMPMuscle-derived signals are essential for both developing and mature motor neurons. Although the target dependence only exists until early postnatal stage (P7 to P10) for the survival of developing motor neurons [27], mature motor neurons continue to receive some neurotrophic signals from adult muscle fibers. For instance, adult motor neurons receive muscle derived neurotrophin-4 (NT-4) for nerve growth and remodeling, although the expression of NT-4 in muscle fibers requires signals from motor neurons [28]. Equally, the disappearance of BMP4 immunoreactivity in muscle fibers following nerve transaction may suggest that BMP4 expression is regulated by motor neurons in a fashion similar to that of NT-4 (Fig. 2). In addition to agrin, we speculate that other unknown factors may be involved in this process. The neuronal regulation of BMP4 may reflect its function in the regulation of neuron survival, axon growth, and maintenance of synaptic connections as reported for NT-4 previously [28,29]. Although it is not clear how agrin regulates BMP4 expression in muscle cells, the activation of Lrp4/Musk receptor complex by agrin has been shown to stimulate JNK pathways which may further induce BMP4 expression [30,31]. The up-regulation of BMP4 in skeletal muscles after nerve ligation could reflect a similar physiological function for BMP4 to that discussed above. Indeed, it has been shown that enhancedFigure 5. Expression of BMP4 mRNA in the neuromuscular system following nerve ligation. Total RNA was isolated from sciatic nerve (n = 4), soleus muscle (n = 6) and lumbar spinal cord (n = 6) of control and nerve-ligated mice. Expression levels of BMP4 mRNA were measured by real-time PCR. Data are presented using GAPDH expression level as 100 . Values are mean 6 SEM (*, P,0.05, compared to normal mice using Student’s t test). doi:10.1371/journal.pone.0058441.gBMP4 and Motor NeuronFigure 6. BMP4 promotes embryonic motor neuron survival and protects NG108-15 neurons against glutamate (Glu)-induced excitotoxicity (Extt). (A) A dose-response curve for the survival effect of BMP4 on embryonic motor neurons. The numbers of surviving motor neurons were determined by counting islet-1 positive neurons under a microscope. Data are presented using cell counts in the vehicle group as 100 . (B ) Differentiated NG108-15 cells were treated with glutamate and/or BMP4 for 16 hours, followed by an 8-hour recovery period in conditioned medium with or without BMP4. Three representative photos show the morphology of differentiated NG108-15 neurons in control (C), glutamate-treated (D) and glutamate plus BMP4-treated (E) conditions. Scale bars = 100 mm. The numbers of surviving neurons were counted under a microscope by.