Genetic Dissection of miR-8 Regulation in Drosophila Neuromuscular Junction

All stocks were maintained and crossed at 25°C according to standard procedures. Stocks were obtained from the Bloomington Stock Center unless otherwise specified. The following loss-of-function alleles and transgenic lines were used: GMR-Yan^Act (ref. 15 (link), 26 (link)) was a gift from R.W. Carthew, UAS-FP₄-mitoEGFP (ref. 23 (link)) was a gift from M. Peifer, ena²¹⁰ and UAS-ena (ref. 27 (link)) were gifts from F. M. Hoffmann, miR-9a^J22 and miR-9a^E39 (ref. 14 (link)) was a gift from F.B. Gao; miR-8-GFP Sensor (ref. 21 (link)) was a gift from H. Ruohola-Baker, EP-atro, and P{EPgy2}GugEY14339. miR-8^Δ2 (ref. 16 (link)) was a gift from S. Cohen; an independent miR-8 null allele ΔmiR-8 was generated as part of a separate study in our laboratory (C.S.L., C.M.L. and D.VV. unpublished observations), tubulinEGFP nerfin-1 3'UTR reporter^{12 (link)} was a gift from J. Brennecke. For the analysis of miR-9a activity in wing imaginal discs, the tubulinEGFP nerfin-1 3'UTR reporter was recombined with ptc-Gal4 on the second autosomal chromosome. The following Gal4 drivers were used to drive ubiquitous, eye, leg, wing disc, pan-neural and mesodermal expression: tubulin-Gal4, GMR-Gal4 and eyeless-Gal4, Dll-Gal4, ptc-Gal4, elav-Gal4 and how^24B-Gal4 respectively. The miR-8-Gal4 line was obtained from the Drosophila Genetic Resource Center (DGRC) and used to drive expression of UAS-CD8GFP.
To generate the allelic combination of ena heterozygous/ΔmiR-8 homozygous genetic background, the ena²¹⁰ allele was recombined with ΔmiR-8 allele on the second autosomal chromosome and the miR-8 NMJ phenotypes were assessed as described above. To test the effect of postsynaptic Ena inhibition on ΔmiR-8 induced NMJ phenotype, UAS-FP₄-mito was expressed using the how^24B-Gal4 driver, in a ΔmiR-8 homozygous mutant background. The specificity of the UAS-FP₄-mito has been previously described^{23 (link)}.