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Figure 2 | Neural Development

Figure 2

From: Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways

Figure 2

BMP-evoked Smad1/5/8 phosphorylation, but not collapse activity, depends on type I BMP receptor kinase activity. (A) Whole cell lysates of dissociated dI neurons with or without DM (10 μM) and with or without BMP7 or BMP6 (50 ng/ml), probed and normalized on western blots as in Figure 1B. Results are expressed as percentage of control pSmad1/5/8 for each condition (BMP7 - DM = 188 ± 28%; BMP7 + DM = 104 ± 3%; BMP6 - DM = 191 ± 25%; BMP6 + DM = 122 ± 13%; n = 2). DM had no effect on baseline pSmad1/5/8 (control + DM = 100 ± 13%). pSmad1/5/8 responses to BMP7 and BMP6 treated with DM differ significantly from pSmad1/5/8 responses to BMP7 and BMP6 without DM (*P < 0.05, Mann-Whitney test). (B) Left: pSmad1/5/8 in dissociated dI neurons with or without BMP7 and with or without DM. Scale = 80 μm. Insets: pSmad1/5/8-positive and -negative neurons (scale = 10 μm). Right: the percentage of pSmad1/5/8+ neurons in control cultures with or without DM was unchanged (control - DM = 5.4 ± 4.7%; control + DM = 4.5 ± 4.1%; P = 0.8977, Student's t-test). The number of pSmad1/5/8+ cells in response to BMP7 with or without DM differed significantly (BMP7 - DM = 53.1 ± 1.2%; BMP7 + DM = 4.7 ± 1.8%; **P < 0.005, Student's t-test). Results are mean ± SEM for four 20 × fields: 150 to 500 neurons/condition/experiment (n = 2). (C) ERM-labeled dissociated dI neuron sister cultures, with or without BMP7 and with or without DM as in (B). The growth cone area in control cultures was not changed by DM (not shown; control - DM versus control + DM, P = 0.9215 (Student's t-test)). The percentage decrease in growth cone area in response to BMP7 was not significantly altered by DM: control + DM = 1.3 ± 11%; BMP7 - DM = 36 ± 4%; BMP7 + DM = 32 ± 2%; n = 2; BMP7 - DM versus BMP7 + DM, P = 0.4097 (Student's t-test)). Arrowhead indicates control growth cone and asterisks indicate collapsed growth cones. Scale = 10 μm.

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