We noted that 30%–40% of motor neurons are preserved in laterally located LMC motor pools in the absence of GDE2 at E13.5. This number is remarkably similar to that reported for the gamma motor neuron component of motor pools, which are predicted to begin diversifying from alpha motor neurons by E13.5, given their differential sensitivities to embryonic programmed cell death (Burke et al., 1977, Friese et al., 2009, Buss et al., 2006 and Hui et al., 2008). To examine whether GDE2 selectively

regulates the differentiation of alpha, but not gamma, motor neurons, we compared Gde2−/− animals with WT siblings at postnatal day 5 (P5) and P28, when molecular and somal size differences allow alpha and gamma motor neurons to be distinguished ( Friese et al., 2009). The percentage GSI-IX ic50 of ChAT+/NeuN+ alpha motor neurons in the ventral outer quadrant of the spinal cord corresponding to the LMC was decreased by approximately 30%–40% at P5 and P28 in Gde2−/− animals; however, the percentage

of ChAT+/NeuN− gamma motor neurons was not significantly altered ( Figures 5A–5F). The expression of Err3 in the ventral horn of the spinal cord appeared to be similar between Gde2−/− and WT littermates, consistent with preserved gamma motor neuron differentiation in the Pifithrin-�� absence of GDE2 ( Figures 5G and 5H). Gamma motor neurons have a small somal area compared with alpha motor neurons ( Burke et al., 1977, Friese et al., 2009 and Shneider et al., 2009). The number of putative gamma motor neurons (somal area < 380 μm2) was unchanged between WT and Gde2−/− littermates, but there was a dramatic reduction of putative alpha motor neurons in Gde2−/− animals (somal

area = 380–1,400 μm2) ( Figures 5I and 5J). Using the same criteria discussed above, no significant changes in alpha and gamma motor neuron numbers were observed in the medially located MMC of Gde2−/− and WT animals ( Figures 5K–5O). Thus, the reduction in LMC motor pools in Gde2 null animals correlates with Megestrol Acetate a specific loss of alpha motor neurons, whereas LMC gamma motor neurons and MMC alpha and gamma motor neuron production are intact. At hindlimb levels, GDE2 is first localized to motor neuron cell body areas at the time of motor neuron generation but is subsequently enriched in motor axons from E12.5 (Figure 6B; Figure S5). To define when GDE2 functions in hindlimb motor pool formation, we generated Gde2lox/−; Rosa26:CreER+ animals, which enabled the timed ablation of GDE2 through Cre-dependent recombination via the administration of 4-hydroxytamoxifen (4-OHT) ( Badea et al., 2003). We injected pregnant dams with 4-OHT at E8.5 to ablate GDE2 expression prior to the initiation of motor neuron progenitor differentiation at lumbar levels and at E10.5 to eliminate GDE2 by the end of motor neuron generation ( Nornes and Carry, 1978).