Fig. 2. grain is required for ISN motor axon projections. Stage 16 embryos stained with -Fas2 (green in A-C,G), RN2-GAL4 driving UAS-mEGFP^F (green in D-H), CQ2-GAL4 driving UAS-mEGFP^F (green in I,J) and Phalloidin-TX (magenta in A-F,H-J). Arrows and arrowheads indicate axons terminals contacting dorsal (2 and 10) and dorsal-most (1 and 9) muscles, respectively. (A) In wild type, the ISN nerve innervates muscles 2/10 and 1/9. (B) In grn mutants, ISN fails to innervate muscles 1/9, but axonal projections are seen contacting muscles 2/10. Bracket denotes a partially penetrant muscle patterning phenotype, evident as an imprecise insertion of muscles 21-24 into the body wall. (C) In grn rescue (RN2-GAL4/UAS-grn; grn^-/-) ISN innervates muscles 2/10 and 1/9 as in wild type. (D) In control, RN2-GAL4/UAS-EGFP^F reveals muscle 1 innervation by aCC and muscle 2 innervation by RP2. (E) In a grn mutant background, RN2-GAL4/UAS-EGFP^F reveals that although muscle 1/9 is not innervated by aCC, axon terminals from aCC and/or RP2 contact muscles 2/10. (F) zfh1 can partially rescue grn mutants (RN2-GAL4/UAS-zfh1; grn^-/-) and the lack of muscle 1 innervation (arrowheads) is less severe than in grn mutant. (G) Overlap between RN2-GAL4/UAS-EGFP^F (green) and -Fas2 (magenta) revealing axons terminals for aCC and RP2. This reporter allows for a precise analysis of aCC and RP2 terminals in the periphery. (H) In grn mutants, 36% of hemisegments (n=69) show ectopic innervation of muscle 8 together with defasciculation of aCC and RP2 motor axons (see also oblique arrow in E). (I) In control, CQ2-GAL4/UAS-EGFP^F reveals muscle 9 innervation by U1 and muscle 10 innervation by other U motoneurons. (J) In a grn mutants, CQ2-GAL4/UAS-EGFP^F reveals that muscle 9 is not innervated (by U1), while U axon terminals contact muscles field 2/10. (K) Quantification of muscles 1/9 innervation in different genetic backgrounds. (L,M) Schematic showing the grn mutant phenotypes (M) compared to wild type (L).