Figure 2. Mutations in hts result in severe synaptic retractions.

A-D) NMJs stained with markers for the presynaptic active zone protein Brp (green), postsynaptic glutamate receptors (DGluRIII, red) and the presynaptic membrane (HRP, white). The insets show individual channels at higher magnifications.

A) Wild type (w¹¹¹⁸) muscle 4 NMJ, the presynaptic active zone protein Brp is found in perfect apposition to postsynaptic glutamate receptors.

B) A hts^W532X/hts^ΔG mutant muscle 4 NMJ. Two motoneurons innervate the muscle. One NMJ (arrow) lacks all presynaptic Brp staining. The presynaptic membrane is fragmented and no longer continuous with the main motoneuron nerve. At higher magnification it becomes evident that the glutamate receptor clusters appear fused and are no longer organized into distinct uniform clusters.

C) A Df^BSC26/hts¹¹⁰³ mutant muscle 4 NMJ. Only very small amounts of presynaptic Brp can be detected that are no longer organized into distinct puncta (asterisk). In addition, the presynaptic membrane is fragmented and discontinuous (arrow) indicating the retraction of synapses.

D) A Df^BSC26/hts¹¹⁰³ mutant muscle 6/7 NMJ. At large parts of the NMJ the presynaptic marker Brp is absent although postsynaptic glutamate receptors are still present. The presynaptic membrane is fragmented at these sites (arrows). Scale bar in A applies to A-D and represents 10 μm; insets 2 μm.

E) Quantification of the frequency of synaptic retractions. All combinations of hts mutants show a significantly higher number of retractions compared to wild type (n > 10 animals and > 100 NMJs each; *** indicates p < 0.001; * indicates p < 0.05, ANOVA). We do not observe significant numbers of retractions in hts^W532X/w¹¹¹⁸ mutant animals demonstrating that the observed phenotypes in hts^W532X/hts^ΔG are in part due to the lack of the MARCKS domain in the hts^ΔG mutation.

F) Quantification of the severity of synaptic retractions. The severity was quantified based on the number of postsynaptic bouton profiles that lack the presynaptic marker Brp. Numbers represent percentage of total NMJs scored (n as in E). Elimination indicates the complete absence of distinct Brp signal and the complete fragmentation of the presynaptic membrane from one motoneuron innervation site (examples in B and C). Error bars represent SEM.