, 2005, Jaworski and Burden, 2006 and Li et al., 2008). Moreover, when β-catenin is ablated in muscle cells using HSA-Cre, mutant mice die immediately after birth without functional NMJs (Li et al., 2008). We further generated double knockout mice—HSA-Cre;HB9-Cre;LRP4f/f (HSA/HB9-LRP4−/−)—in which the LRP4 gene is ablated in both motoneurons and muscles. Remarkably, HSA/HB9-LRP4−/− pups died soon after birth with cyanosis. AChR cluster formation was severely

impaired (Figure 4) as clusters were almost undetectable in HSA/HB9-LRP4−/− diaphragms, except occasional smaller, weak clusters. Their CH5424802 cost size was only 9.6% of that in LRP4loxP/+ control and 34.9% of that in HSA-LRP4−/− (Table S1). Quantitatively, the number of AChR clusters in double KO pups was reduced by 95.5%, compared to LRP4loxP/+ controls, and by 96.2%, compared to HSA-LRP4−/− pups (588 ± 95.9 per mm2 in controls, 707 ± 89.2 per 3-Methyladenine mouse mm2 in HSA-LRP4−/−, and 26.7 ± 15.8 per mm2 in HSA/HB9-LRP4−/− pups) (Table S1). These results demonstrate that the ablation of LRP4 in motoneurons further impairs AChR clustering in HSA-LRP4−/− mice and identifies a role of motoneuron LRP4 in AChR clustering (Figure 4F). As observed in LRP4mitt null mice, aneural AChR clusters were almost undetectable in diaphragms of E13.5 HSA/HB9-LRP4−/− embryos, indicating impaired prepatterning of muscle fibers (Figure S4A). The presynaptic deficits in HSA/HB9-LRP4−/−

mice also resemble those in LRP4mitt null as the number and length of secondary and tertiary branches of these two genotypes were similar (Table S1) (Figures 4A and 4B).

However, compared to HSA-LRP4−/−, secondary branches were significantly longer in HSA/HB9-LRP4−/− and LRP4mitt null mice, indicating a role of motoneuron LRP4 in nerve terminal differentiation. This notion was supported by increased number of tertiary and quaternary branches in HSA/HB9-LRP4−/− and LRP4mitt mice (Figure 4E). Moreover, motor nerve terminals appeared to be fragmented in diaphragms of both HSA/HB9-LRP4−/− mice and LRP4mitt null mice. In contrast, such discontinuous intumescence of nerve terminals was not observed in HSA-LRP4−/− muscles (Figures S4B and S4C). LRP4 null mutation or conditional mutation (in muscles or in both muscles and motoneurons) had little during effect on the number and distribution of motoneurons (Figures S4D and S4E), suggesting that LRP4 controls neuron differentiation, but not survival. To investigate how muscle LRP4 regulates presynaptic differentiation, we tested whether LRP4 could be synaptogenic using an established coculture assay (Biederer et al., 2002, Fogel et al., 2011, Graf et al., 2004 and Scheiffele et al., 2000). HEK293 were transfected with EGFP alone (control) or together with LRP4 and cocultured with cortical neurons. Cells were stained for synapsin and SV2, both markers for presynaptic differentiation.