Figure 3. Lar/sns transheterozygotes have the same phenotypes as Lar mutants and Sns knockdowns.

Neuromuscular junctions (NMJs) were analyzed using a published Fiji macro (Nijhof et al., 2016) that uses HRP to outline boutons and measures NMJ area, perimeter, length, longest branch length, number of branches, number of boutons, and Bruchpilot (Brp) labeled punctae. (A–F) Representative images of the NMJ on muscles 7/6 from WT and heterozygote controls (A–C), Lar/sns transheterozygotes (D, E), and Lar mutants (F). NMJs are labeled with anti-HRP (red). NMJ outlines showing boutons and branch architecture as outputs from the macro are under each NMJ image. (G–K) Quantification of 1b NMJ parameters, showing reduced NMJ size and arborization in Lar/sns transhets and Lar mutants (red) compared to het controls (blue). Data is average from segments A2–A4 from minimum 30 NMJs per genotype. (L, M) Quantification of Brp punctae showing reduced number of active zones in Lar/sns transhets and Lar mutants. (N–P) Representative images of NMJs on muscles 7/6 from animals with RNAi-mediated neuronal knockdown of Lar and Sns. Neuronal Lar or sns RNAi results in the same NMJ abnormalities seen in genetic Lar/sns transhets and Lar mutants. (Q–S) Quantification of NMJ parameters showing reduced 1b NMJ area, number of boutons, and NMJ length upon either Lar or Sns knockdown. A2–A4 segments were analyzed from at least 30 NMJs on muscles 7/6. All datasets were analyzed using one-way ANOVA followed by Tukey’s post-hoc correction. ****p<0.0001; ***p<0.001. Scale bar, 20 µm. See Figure 3—figure supplement 1 for analysis of 1s NMJs.

Figure 3—figure supplement 1. 1s neuromuscular junction (NMJ) abnormalities in Lar/sns transhets.

NMJs were analyzed using a published Fiji macro (Nijhof et al., 2016) that uses HRP to outline boutons. NMJ area, length, and number of boutons were quantified specifically for 1s boutons. (A–F) Representative images of the NMJ on muscles 7/6 from control (A–C) and transhet (D–F) animals. NMJs are labeled with HRP (red). NMJ outlines showing 1s boutons and branch architecture as outputs from the Macro are under each NMJ image. (G–I) Quantification of 1s NMJ parameters showing reduced NMJ size and boutons in Lar/Sns transhets and Lar mutants (red) compared to het controls (blue). Data is average from segments A2–A4 from minimum 30 NMJs per genotype. All datasets were analyzed using one-way ANOVA followed by Tukey’s post-hoc correction. ****p<0.0001. Scale bar, 20 µm.

Figure 3—figure supplement 2. 1b neuromuscular junction (NMJ) abnormalities in Lar/sns transhets, Sns RNAi in Lar mutants, and neuronal Kirre RNAi.

1b boutons on the 7/6 NMJ were analyzed as described. (A–E) Quantification of 1b NMJ parameters: (A) NMJ area, (B) number of 1b boutons, (C) total NMJ length, (D) longest 1b branch length, and (E) number of 1b branches. Lar^13.2/sns^Df transhets show similarly reduced NMJ parameters as observed with other Lar/sns transhets. Addition of sns RNAi to Lar null mutants does not increase the severity of the Lar mutant phenotype. Neuronal knockdown of Kirre does not cause 1b NMJ abnormalities. Data is average from segments A2–A4 from minimum 30 NMJs per genotype. All datasets were analyzed using one-way ANOVA followed by Tukey’s post-hoc correction. ****p<0.0001, **p< 0.01.