Fig. 7: Muscle fiber-specific knockout of Pdzrn4 causes NMJ fragmentation.

a Adult Cas9mKI mice were injected with AAVMYO encoding 6 (Lrtm1) or 7 (Etv4, Pdzrn4 and Pdzrn3) different sgRNAs (3 × 10¹¹ vector genome copies) via the tail vein. Mice injected with Lrtm1 or Pdzrn4 sgRNAs were female and mice injected with Etv4 sgRNAs were male. b Relative mRNA expression, measured by qPCR, of the targeted genes in TA muscle 15 weeks post-injection. Note that Musk mRNA was also significantly reduced in all three groups. c All-limb and forelimb grip strength measurements were performed on mice 10 weeks post injection. d CMAP was performed on the GAS 15 weeks post-injection at different frequencies (image shows average values at 40 Hz frequency). e, f, and g representative whole-mount images of NMJs from the EDL from Etv4 (e), Lrtm1 (f), and Pdzrn4 (g) knockout (KO) mice and control littermates. AChRs are visualized by α-bungarotoxin and the presynapse by neurofilament (NF) and synaptophysin (SYP). Bar graphs next to the images show the quantification of AChR fragmentation. NMJs of Pdzrn4 KO mice are significantly fragmented. h Relative mRNA expression of the targeted genes in TA muscle 15 weeks post-injection of AAVMYO encoding sgRNA to Pdzrn3 or Pdzrn4. Double knockout for Pdzrn3 and Pdzrn4 (Pdzrn3/4 dKO) was achieved by co-injection of the two AAVMYOs. i Representative whole-mount images of NMJs from the EDL from Pdzrn3/4 dKO and control mice. j AChR clusters/NMJ were counted and quantified. Unlike the single Pdzrn4 KO, both Pdzrn3 KO and the Pdzrn3/4 dKO show no fragmentation of the postsynapse. The experimental scheme in (a) was created in BioRender. The data in (b-j) is presented as mean ± SEM. In (b–j) a two-tailed t-test was performed. p < 0.05 = *p < 0.01 = **p < 0.001 = ***. For whole-mount quantifications, a minimum of 10 NMJs per mouse were quantified (n = 4 for Etv4, Lrtm1, Pdzrn3, Pdzrn3/4 dKO and n = 5 for Pdzrn4 KO). Source data and p-values are provided as a Source Data file.