5 but is substantially decreased by E12.5 ( Figures 1A–1D; data not shown). Consistent with previous studies showing a requirement for GDE2 in interneuron generation, Gde2 transcripts extend dorsally from E10.5, coincident with the timing of ventral and dorsal interneuron formation ( Figures 1B and 1C; Yan et al., 2009). Similarly, GDE2 protein is expressed in postmitotic somatic motor neurons from E9.5 and is detected dorsally from E10.5 ( Figures 1E and 1F). Examination of GDE2 expression in relation to columnar-specific

motor neuron markers at fore- and hindlimb levels of the spinal cord shows that GDE2 is localized to newly differentiating motor neurons and to MMC and lateral and medial LMC motor neurons ( Figures 1E–1G’; Tsuchida et al., 1994; data not shown). By E12.5, GDE2 protein is reduced within motor neuron check details cell bodies but is enriched within motor axons, suggesting that GDE2 may have later roles in postmitotic motor neuron development ( Figures 1H and 1I). Thus, GDE2 is expressed in somatic motor

neuron cell bodies coincident with the period of motor neuron neurogenesis. To test the requirement for GDE2 in regulating motor click here neuron generation, we generated stable mouse lines that lack functional GDE2 (Gde2−/−) using Cre-lox technology (see Figure S1 available online). We confirmed GDE2 ablation using a combination of PCR, direct sequencing, western blot, aminophylline and immunohistochemical analyses ( Figure 7C; Figure S1). Examination of Gde2−/− and wild-type (WT) littermates at the onset of motor neuron differentiation at E9.5 showed an approximately 50% loss of Isl1/2+ and HB9+ motor neurons ( Figures 2A, 2B, 2D, 2E, and 2G; Nornes and Carry, 1978). However, the number of Olig2+ motor neuron progenitors and the dorsal-ventral patterning of spinal progenitors were not affected ( Figures 2C, 2F, and 2G; Figure S2). No increase in TUNEL

staining was detected in Gde2−/− animals, suggesting that the loss of GDE2 does not compromise motor neuron survival but instead disrupts motor neuron formation ( Figure S2). Consistent with this model, Gde2 null mutants showed a decrease in the number of progenitors exiting the cell cycle ( Figures 2J, 2M, and 2N). Although no changes in the proportion of cells in S phase and M phase were detected, the total number of cells in S phase after a 16 hr BrdU pulse was increased, suggesting that the length of the cell cycle is extended in the absence of GDE2 ( Figures 2H–2N; Yan et al., 2009). These data collectively support previous findings in the chick showing that GDE2 is required to regulate motor neuron generation but does not affect progenitor patterning and specification ( Rao and Sockanathan, 2005). Some motor neurons are generated in the absence of GDE2, suggesting that GDE2 function might be redundant with its family members Gde3 and Gde6 ( Nogusa et al., 2004 and Yanaka et al., 2003).