We focused on the genes that are misregulated in Bhlhb5 mutant mice at the time of the axon targeting defects (i.e., from E13.5 to E17.5). One of these genes is Cdh11, a classic type II cadherin that mediates homophilic cell-cell adhesion ( Kimura et al., 1995). Cdh11 mRNA is expressed in differentiating neurons of the cortical plate, including layer V projection neurons that form the corticospinal tract ( Kimura

et al., 1996). Furthermore, we found that Cdh11 protein is highly expressed in the axons of corticofugal neurons at E16.5, when these projection neurons are extending their projections through the internal capsule ( Figure 7C, white arrows), consistent with the idea that Cdh11 PD-1 inhibitor may play a role in their guidance. In addition, the subcortical expression pattern of Cdh11 is

suggestive of a possible role in regulating the connectivity of corticospinal motor neurons. In particular, Cdh11 is specifically expressed in a number of intermediate subcortical targets where corticospinal motor neurons form collaterals, namely the red nucleus, the basilar pons, and the inferior olive ( Kimura et al., 1996). Importantly, Bhlhb5 and Anti-diabetic Compound Library Prdm8 bind to an intron within the Cdh11 gene ( Figures 5D, 5G, and 5J) and Cdh11 mRNA is upregulated in both Bhlhb5 and Prdm8 mutant mice during embryonic development ( Figures 7A and 7B). Upon loss of Bhlhb5, the overall level of Cdh11 protein appears elevated, whereas the pattern of Cdh11 expression is unaffected ( Figures 7C and 7D), suggesting that a Bhlhb5/Prmd8 repressor complex may function to restrain the level of Cdh11 rather than its distribution. Based on these observations, we hypothesized that Cdh11 might be a target of the Bhlhb5/Prdm8 repressor complex whose upregulation in the absence of Bhlhb5 or Prdm8 leads to a disruption of the formation of the corticospinal these tract. Specifically,

overexpression of Cdh11 in axons of corticospinal motor neurons might impede their progress due to enhanced adhesion to Cdh11-expressing intermediate targets. This might then prevent these Cdh11-overexpressing axons from extending past Cdh11-expressing intermediate targets and into the spinal cord. If so, we reasoned that reducing the level of Cdh11 in Bhlhb5 mutant mice might at least partially rescue the axon extension defects in corticospinal motor neurons. To test this idea, we obtained Cdh11 mutant mice (Cdh11−/−), which lack functional Cdh11 due to a targeted disruption of the extracellular domain and most of the transmembrane domain ( Horikawa et al., 1999). Importantly, mice lacking Cdh11 show normal targeting of corticospinal axons ( Figure 7G; data not shown).