Fig. 5: Regulation of Hts-M isoforms in muscle accounts for miR-34 promotion of bouton addition.

a, b MARCKS domain-containing Hts-M protein isoforms (green) surround presynaptic boutons stained with anti-HRP (magenta) at the M6/7NMJ. Hts-M signal is observed in the muscle with high levels surrounding type 1 boutons in w¹¹¹⁸ control (a) and miR-34 null (b) NMJs (segment A2; Scale bar: 10 µm). High magnification insets separating the HRP (a′, b′) and Hts-M (a″, b″) channels (merged in a‴, b‴) reveal increased Hts-M signal in and beyond the SSR halo surrounding large boutons in miR-34 mutants. c Fluorescence intensity profiling of Hts-M antigen within a region of interest surrounding the NMJ and adjacent muscle (see Methods) reveals a significant increase in immunolabeling (* p ≤ 0.05). d Alignment of the conserved MRE for Human (hsa) miR-34a within the 3′UTR of Adducin2 (Add2) is shown in comparison to the corresponding MRE for Drosophila (dme) miR-34 in the Hts 3′UTR downstream of the MARCKS domain coding sequence. e Muscle-specific competitive inhibition of miR-34 using DMef2-GAL4 to express miR-34SP compared to Hts-M isoform overexpression (Hts-M OE via UAS-hts-M^wtVK33) using the same DMef2-GAL4 driver (left and right red bars), reveals highly significant deficits in type 1 boutons that are not significantly different from the decrease observed in miR-34SP (p = 0.79). However, motor neuron-specific OK6-GAL4 driven Hts-M OE fails to induce a significant change in bouton number (gray bar); n = 20 NMJs for each genotype including control lines which differences are graphed relative to. f DMef2-GAL4 expression of miR-34SP induces a significant decrease in type 1 bouton numbers per NMJ (red bar) compared to Scramble-SP controls (white bar); this phenotype is completely rescued by co-expression of UAS-hts^RNAi (gray bar) n = 20 NMJs for each genotype. g Using Mhc-GAL4 and the same UAS-hts^RNAi transgene, we also observe effective rescue of the miR-34 null type 1 bouton phenotype; n = 20 for Control, n = 19 for miR-34 Null, n = 20 for Muscle Rescue. h Parallel experiments using spaced depolarization to induce activity-dependent nascent boutons show that controls (w¹¹¹⁸ above, DMef-GAL4 below) show highly significant increases after K+ treatment compared to mock-treated samples (green bars), whereas, both miR-34 null (above) and UAS-hts-M^VK33 over- expression (OE, below) produce no induction (gray bars). For c and e–h: Error bars indicate ±SEM; **p-value ≤ 0.01; ***p-value ≤ 0.01; ns is p > 0.05; For h upper panel: w¹¹¹⁸ and miR-34 mock n = 13 for, K+ n = 9, w¹¹¹⁸ mock n = 8, K+ n = 11; For h lower panel: DMef-GAL4 experiments HTS-M OE mock n = 24, K+ treated n = 18, control mock n = 13, K+ treated n = 11. i A working model of miR-34 action in the presynaptic (green) and postsynaptic compartments of type 1 boutons showing how regulation of Nrx-IV in motor neurons can influence active zones while regulation of Hts-M isoforms controls formation of nascent boutons, possibly via modulation of remodeling of SSR and/or synaptic adhesion. Source data are provided as a Source Data file.