To determine whether GDE2 is required for the generation of motor neurons of distinct subtypes, we examined motor column formation in WT and Gde2 null littermates ( Figure S3; Tsuchida et al., 1994, Rousso et al., 2008 and Dasen et al., 2008). At fore- and hindlimb levels, Gde2 null animals showed an approximately 40%–50%

loss of medial and lateral LMC neurons at E11.5 and a decrease of 30%–35% at E13.5 ( Figures 3A–3G and 3O–3U), whereas we noted a modest decrease of 20% in thoracic HMC neurons at E11.5 ( Figures 3H–3N). Strikingly, no changes in the numbers of MMC neurons or PGC neurons were found at either time point ( Figures 3A–3U). The loss of HMC and LMC neurons in Gde2−/− animals is unlikely to be due to impaired Hox activities because of the following: (1) gain or loss of Hox gene function does not reduce motor neuron numbers; (2) expression see more of FoxP1, a critical cofactor of Hox function,

in existing motor neurons is unaffected by the loss Lonafarnib research buy of GDE2 ( Figures 3A–3T; Rousso et al., 2008 and Dasen et al., 2008); and (3) brachial Hoxc6, thoracic Hoxc9, and lumbar Hoxa10 expression are preserved in motor neurons of Gde2−/− animals ( Figure S3; Jung et al., 2010). V2 interneurons derive from Lhx3+ progenitors, and V2 interneuron differentiation programs are actively suppressed in motor neurons by the transcription factor HB9 ( Arber et al., 1999, Thaler et al., 1999 and Thaler et al., 2002). Islet1/2 motor neurons did not coexpress Chx10, no increases in cell death by TUNEL were detected, and V2 interneuron numbers were unchanged in the absence of GDE2, arguing against the possible conversion of prospective HMC and LMC neurons to V2 fates ( Figures S2 and S3; data not shown). Taken together, these observations suggest that GDE2 function is restricted to the formation of LMC and HMC motor neurons and invokes the existence of other regulatory modules that control the formation of GDE2-independent motor neurons. The loss of fore- and hindlimb LMC neurons in Gde2−/− animals indicates that GDE2 activity is not restricted to a specific rostral-caudal domain, whereas the partial reduction

of medial and lateral LMC neurons suggests that GDE2 might be required for the formation of distinct LMC motor pools ( Figure 3). We analyzed Gde2 null L-NAME HCl animals at lumbosacral segment (LS) 2 of the spinal cord, where combined molecular and axonal tracing approaches have defined a molecular code that distinguishes seven medial and lateral LMC motor pools that innervate major muscle groups in the hindlimb ( Figure 4C; De Marco Garcia and Jessell, 2008, Lin et al., 1998 and Arber et al., 2000). These include five motor pools within the medial LMC that innervate the adductor longus and magnus (Al, Am), the adductor brevis (Ab), and the anterior and posterior gracilis muscles (Ga, Gp), as well as two lateral LMC pools that target the vasti (Va) and the rectofemoratibialis muscles (Rf).