5 or E10.5 showed an equivalent loss of GDE2 in motor neuron cell bodies and axons at E12.5, demonstrating that Cre-mediated loss of GDE2 in both cases had occurred prior to detectable LS2 motor pool formation ( Figures 6B, 6D, 6F, and 6H; Figure S5). Analysis of the Va, Al, Am, and Gp motor pools in Gde2lox/−; Rosa26:CreER+ embryos after 4-OHT injection at E8.5 showed a loss
of Isl1/2+ motor neurons and a dramatic reduction of ER81+ Va motor neurons at E12.5 and E14.5 compared with Gde2lox/− and Gde2+/−; Rosa26:CreER+ controls ( Figures 6I–6Q; data not shown). Consistent with the phenotype of Gde2 null animals, Al, Am, and Gp pool http://www.selleckchem.com/products/Staurosporine.html formation was delayed such that a decrease in Er81/Isl1+
motor neuron numbers at E12.5 was mitigated by E14.5 ( Figures 6I–6Q). Thus, elimination of GDE2 prior to the initiation of motor neuron generation mimics the phenotype observed in check details Gde2 null animals. In contrast, administration of 4-OHT at E10.5 did not alter the number of Va, Al, Am, or Gp motor neurons in Gde2lox/−; Rosa26:CreER+ embryos compared with Gde2lox/− and Gde2+/−; Rosa26:CreER+ controls, although the level of GDE2 ablation was equivalent in both cases ( Figures 6F, 6H, 6K, 6L, 6O, 6P, and 6R; Figure S5). These results suggest that GDE2 removal at the onset of neurogenesis disrupts the formation of specific motor pools, whereas GDE2 ablation after motor neuron generation is complete does not. Thus, the ability of GDE2 to regulate the formation
of specific LMC motor pools coincides precisely with the temporal profile of motor neuron neurogenesis and the localization of GDE2 within motor neuron cell bodies and dendrites. To determine how GDE2 regulates motor neuron differentiation, we considered the possibility that GDE2 might downregulate Notch signaling, a pathway known to be required for the maintenance of Olig2+ motor neuron progenitors in an undifferentiated state (Marklund et al., 2010). To test this hypothesis, we compared the expression of two direct downstream targets of activated Notch in Gde2−/− spinal cords in relation to WT littermates. Gde2−/− animals showed a marked expansion of Hes5 and Blbp expression ( Figures 7A, 7B, 7D, and 7E); further, GDE2 ablation Dipeptidyl peptidase increased the amount of Notch intracellular domain (NICD) in dissected ventral spinal cords, in accordance with elevated levels of ligand-dependent Notch processing and an increase of activated Notch signaling ( Figure 7C; Peng et al., 2007). These data collectively suggest that GDE2 is necessary to downregulate Notch signaling in neighboring motor neuron progenitors. To determine whether GDE2 is sufficient to inhibit Notch signaling, we utilized a gain-of-function approach using in ovo electroporation of embryonic chick spinal cords.