Genetic deletion of CLASP2 impairs NMJs in vivo. (a) The size of synaptic AChR clusters and the number of subsynaptic nuclei per synapse are reduced in Clasp2^−/− muscle. NMJs in soleus muscles of wild-type and mutant animals of equal weights were stained for AChRs (red), the Schwann cell marker S-100 (green), and nuclei with DAPI (blue). Synaptic area was determined from AChR labeling of synapses lying en face (see Materials and methods). Nuclei surrounded by S-100 labeling were considered as terminal Schwann cells (see inset in panel labeled S100/DAPI). The number of subsynaptic muscle nuclei was estimated by subtracting the number of Schwann cell nuclei (marked by asterisks) from the number of nuclei underlying the AChR cluster (marked by arrowheads). Bar, 10 µm. Graphs show mean values of respective parameters ± SEM, n = 47 wild-type and 52 Clasp2^−/− NMJs from 3 muscles of each genotype analyzed (*, P < 0.05; **, P < 0.01, two-sided t test). (b) AChR density and AChR insertion rates are reduced in Clasp2^−/− muscle. To determine AChR densities sternomastoid muscles were saturated with α-BTX–Alexa 594. Bar, 25 µm. Means ± SEM, n = 63 and 72 NMJs from 3 wild-type and 3 Clasp2^−/− muscles analyzed (*, P < 0.05; **, P < 0.01, two-sided t test). To estimate AChR insertion rates sternomastoid muscles were denervated to increase AChR turnover; 7 d later AChRs in superficial endplates were saturated in vivo with α-BTX–Alexa 488. After another 7 d (to allow AChR turnover), endplates were saturated with α-BTX–Alexa 594, and the average intensity of Alexa 488 fluorescence in Clasp2^−/− NMJs was normalized to that in wild-type muscles. Bar, 25 µm. Means ± SEM, 28 wild-type and 25 Clasp2^−/− endplates analyzed (*, P < 0.05; **, P < 0.01, two-sided t test). (c) AChR density is reduced in Clip1^−/−;Clip2^−/− muscle. Analysis similar as in b. Given are means ± SEM, n = 30 wild-type and 31 Clip1^−/−;Clip2^−/− (*, P < 0.05; **, P < 0.01, two-sided t test). Bar, 25 µm.