T subpopulations of motor neurons that rely on different survival factors [10]. Bone morphogenetic proteins (BMPs), the 520-26-3 biological activity largest subgroup within the TGF-b superfamily, were originally identified by theirability to induce bone differentiation [12]. To date, 
more than 20 BMPs have been identified, having diverse biological functions, such as cell proliferation, differentiation, morphogenesis and apoptosis [13,14]. Like other TGF-b superfamily members, BMPs signal through a complex involving both a type I and a type II receptor. In general, the type II receptors control the ligandbinding specificity, while the type I receptors determine which downstream signaling pathway is activated. There are five type II receptors and seven type I receptors [15]. The type II receptors associate with specific subfamilies of the superfamily and are referred to as the TGF-b, BMP, Mullerian inhibiting substance and activin A/B type II receptors. The seven type I receptors are ALK1 to ALK7. It has been shown that BMP signaling is required for normal development of the Drosophila neuromuscular system. Mutations of Glass bottom boat (BMP homolog) and its receptors (Wishful Thinking for the type II receptor and Thickveins for the type I receptor) lead to profound defects at the Drosophila NMJ, such as decreased neurotransmitter release, reduced synaptic size and aberrantBMP4 and Motor Neuronpresynaptic ultrastructure [16?8]. However, the function of BMPs in the mammalian neuromuscular system remains unclear. In this study, we report BMP4 as a peripherally-derived factor that may regulate the survival of motor neurons.Results The type II BMP receptor is associated with the NMJWe have previously shown that BMPRII mRNA and protein were detected in the cell bodies of lumbar spinal motor neurons [19]. We further examined the expression of BMPRII in other parts of the neuromuscular system. In the EDL muscle and soleus muscle (not illustrated), strong BMPRII immunoreactivity was associated with many muscle fibers (Fig. 1A, E and F). To examine whether the BMPRII immunoreactivity was associated with NMJs, sections were also co-stained with BTX to label NMJs. Approximately 71.7 of the BMPRII immunoreactivity was found to overlap with BTX-labeled NMJs (Fig. 1C ). BMPRII immunoreactivity, however, was not detected in sciatic nerves (data not shown).BMP4 mediates motor CAL 120 neuron and muscle interactionsThe association of BMPRII proteins with the NMJs indicated that motor neurons may receive a muscle-derived BMP. We further investigated whether BMP2, BMP4 or BMP6 is a possible candidate for mediating motor neuron and muscle interactions. Differentiated NG108-5 neurons and C2C12 muscle cells were used here because they are capable of forming functional neuromuscular synapses when co-cultured [20]. We found that BMP4 mRNA had a much higher expression level in differentiated C2C12 muscle cells compared to BMP2 and BMP6 (Fig. 2A), and was barely detectable in NG108-15 
neuron cells (Fig. 2B). A lower expression level of BMP4 mRNA was also confirmed in lumbar spinal motor neurons. In the laser capture-isolated motor neuron samples, only a very low level of the BMP4 mRNA could be detected, while BMPRII mRNA as a positive control, showed a much higher expression level (Fig. 2C). These results led us tofurther investigate the function of BMP4 in the neuromuscular system. Localization of the BMP4 protein in the soleus muscle was examined using immunohistochemistry. BMP4-immunoreactivity.T subpopulations of motor neurons that rely on different survival factors [10]. Bone morphogenetic proteins (BMPs), the largest subgroup within the TGF-b superfamily, were originally identified by theirability to induce bone differentiation [12]. To date, more than 20 BMPs have been identified, having diverse biological functions, such as cell proliferation, differentiation, morphogenesis and apoptosis [13,14]. Like other TGF-b superfamily members, BMPs signal through a complex involving both a type I and a type II receptor. In general, the type II receptors control the ligandbinding specificity, while the type I receptors determine which downstream signaling pathway is activated. There are five type II receptors and seven type I receptors [15]. The type II receptors associate with specific subfamilies of the superfamily and are referred to as the TGF-b, BMP, Mullerian inhibiting substance and activin A/B type II receptors. The seven type I receptors are ALK1 to ALK7. It has been shown that BMP signaling is required for normal development of the Drosophila neuromuscular system. Mutations of Glass bottom boat (BMP homolog) and its receptors (Wishful Thinking for the type II receptor and Thickveins for the type I receptor) lead to profound defects at the Drosophila NMJ, such as decreased neurotransmitter release, reduced synaptic size and aberrantBMP4 and Motor Neuronpresynaptic ultrastructure [16?8]. However, the function of BMPs in the mammalian neuromuscular system remains unclear. In this study, we report BMP4 as a peripherally-derived factor that may regulate the survival of motor neurons.Results The type II BMP receptor is associated with the NMJWe have previously shown that BMPRII mRNA and protein were detected in the cell bodies of lumbar spinal motor neurons [19]. We further examined the expression of BMPRII in other parts of the neuromuscular system. In the EDL muscle and soleus muscle (not illustrated), strong BMPRII immunoreactivity was associated with many muscle fibers (Fig. 1A, E and F). To examine whether the BMPRII immunoreactivity was associated with NMJs, sections were also co-stained with BTX to label NMJs. Approximately 71.7 of the BMPRII immunoreactivity was found to overlap with BTX-labeled NMJs (Fig. 1C ). BMPRII immunoreactivity, however, was not detected in sciatic nerves (data not shown).BMP4 mediates motor neuron and muscle interactionsThe association of BMPRII proteins with the NMJs indicated that motor neurons may receive a muscle-derived BMP. We further investigated whether BMP2, BMP4 or BMP6 is a possible candidate for mediating motor neuron and muscle interactions. Differentiated NG108-5 neurons and C2C12 muscle cells were used here because they are capable of forming functional neuromuscular synapses when co-cultured [20]. We found that BMP4 mRNA had a much higher expression level in differentiated C2C12 muscle cells compared to BMP2 and BMP6 (Fig. 2A), and was barely detectable in NG108-15 neuron cells (Fig. 2B). A lower expression level of BMP4 mRNA was also confirmed in lumbar spinal motor neurons. In the laser capture-isolated motor neuron samples, only a very low level of the BMP4 mRNA could be detected, while BMPRII mRNA as a positive control, showed a much higher expression level (Fig. 2C). These results led us tofurther investigate the function of BMP4 in the neuromuscular system. Localization of the BMP4 protein in the soleus muscle was examined using immunohistochemistry. BMP4-immunoreactivity.
