Drosophila let-7-C locus, knockouts, and rescuing transgenes. (A) The Drosophila let-7-C locus, located at cytological location 36E on chromosome 2, encodes a 2435-nt primary transcript containing three evolutionarily conserved miRNAs: miR-100, let-7, and miR-125. (B) The let-7-C^KO1 and let-7-C^GKI mutations contain 1071- and 991-base-pair (bp) deletions respectively, removing miR-100, let-7, and miR-125. The CG10283 gene, located proximally to let-7-C, is also disrupted by the let-7-C^KO1 mutation. The let-7-C^GKI mutation contains Gal4 coding sequences driven by let-7-C transcription (see Supplemental Material for details on let-7-C^GKI strain generation). (C) The P{W8, let-7-C} rescuing transgene includes a 17,983-bp genomic fragment containing the let-7-C locus. Derivatives of P{let-7-C} (not shown) contain 10- to 15-bp deletions removing portions of the mature miR-100, let-7, or miR-125 sequence (see the Supplemental Material for details of P{W8, let-7-C^Δmir-100}, P{W8, let-7-C^Δlet-7}, and P{W8, let-7-C^ΔmiR-125} transgene construction). (D) Expression of miR-100, let-7, and/or miR-125 RNA is eliminated in the let-7-C^KO1, let-7-C^GKI, and let-7-C^KO1/GKI strains and is restored by P{W8, let-7-C} and derivative rescuing transgenes. Samples of total RNA from 1-d-old male flies of the following genotypes were analyzed by Northern blot: wild type in lane 1, let-7-C^KO1/KO1 in lane 2, let-7-C^GKI/GKI in lane 3, let-7-C^KO1/GKI in lane 4, let-7-C^KO1/GKI; P{W8, let-7-C} in lane 5, let-7-C^KO1/GKI;P{W8, let-7-C^Δmir-100} in lane 6, let-7-C^KO1/GKI;P{W8, let-7-C^Δlet-7} in lane 7, let-7-C^KO1/GKI;P{W8, let-7-C^ΔmiR-125} in lane 8, and P{UAS-let-7-C}; let-7-C^KO1/GKI in lane 9. Northern blots were probed for miR-100, let-7, miR-125, and miR-1 RNAs, and miR-1 expression was used as a loading control.

let-7-C is expressed in the nervous system and muscles of adult flies. (A) let-7-C^KO1/GKI flies carrying a UAS-let-7-C transgene are rescued for climbing activity. (Column 1) let-7-C^KO1/GKI; (column 2) let-7-C^KO1/GKI; P{UAS-let-7-C}/+. (B) Ventral section of the VNC of a let-7-C^GKI/+; P{UAS-mCD8-GFP}/+ fly stained for GFP (green) and the neuropil marker nc82 (purple). let-7-C∷Gal4-driven GFP is expressed in neurons in the ventral nerve cord (VNC) and is enriched in the posterior tip of the abdominal ganglion (arrowheads). In all panels, anterior is up. (C) let-7-C∷Gal4 is expressed in neurons that project from the VNC posteriorly into the abdominal cavity. The carcass of a newly hatched let-7-C^GKI/+; P{UAS-mCD8-GFP}/+ male filleted on its dorsal midline and stained for GFP (green) and the F-actin marker rhodamine phalloidin (purple). GFP-positive neurons can be seen running along the nerve cord in each abdominal segment. In the fourth abdominal segment (A4), GFP+ neurons laterally contact the DMs (arrowhead) as well as the DIOMs (asterisk). The white appearance of the DIOMs indicates the colocalization of GFP and rhodamine phalloidin, signifying robust GFP expression. (D) let-7-C∷Gal4 is expressed in motoneurons that innervate the DMs. DMs from a let-7-C^GKI/+; P{UAS-mCD8-GFP}/+ fly stained for GFP (green) and rhodamine phalloidin (purple). The GFP-only channel is also presented, and the neuromuscular junctions of GFP⁺ neurons are indicated (arrowheads). (E) let-7-C∷Gal4 is expressed in the DMs. DMs from a let-7-C^GKI/+; P{UAS-nls-GFP}/P{UAS-nls-GFP} fly stained for GFP (green) and rhodamine phalloidin (purple). The GFP-only channel indicates expression of let-7-C^GKI in muscle cells. Expression of muscle cell GFP is also detectable in D, but is much less obvious. Bars: A–C, 100 μm; D,E, 10 μm.

let-7 miRNA alone is required for the let-7-C-dependent larval-to-adult remodeling of the abdominal neuromusculature. (A–C) Dorsal sections of A4 segment from 2-d-old miR-100^Δ (A), let-7^Δ (B), and miR-125^Δ (C) males stained for rhodamine phalloidin. Note the persistent DIOMs (white asterisks) and small DMs (arrowheads) in let-7^Δ relative to miR-100^Δ and miR-125^Δ. (C,D) Adult let-7 DM neuromuscular junctions fail to achieve wild-type size. DMs from 2-d-old miR-100^Δ (D), let-7^Δ (E), and miR-125^Δ (F) males stained for rhodamine phalloidin (purple) and HRP (green). HRP-only channels are also shown. Bars: C (applies to A–C), 100 μm; F (applies to D–F), 10 μm.

let-7-C is required for normal adult behavior. For all assays, the following genotypes were analyzed: wild type in column 1, let-7-C^KO1/GKI in column 2, let-7-C^KO1/GKI; P{W8, let-7-C} in column 3, let-7-C^KO1/GKI;P{W8, let-7-C^Δmir-100} in column 4, let-7-C^KO1/GKI;P{W8, let-7-C^Δlet-7} in column 5, and let-7-C^KO1/GKI;P{W8, let-7-C^ΔmiR-125} in column 6. For descriptions of the behavioral assays, see the Supplemental Material.

The adult neuromusculature of let-7-C^KO1/GKI mutants display persistent pupal as well as immature adult characteristics. (A,B) Dorsal sections of abdominal segments 3–5 (A3–A5) from 2-d-old wild-type (A) and let-7-C^KO1/GKI mutant (B) males stained for rhodamine phalloidin. The dorsal vessel (dv), which runs in an anterior–posterior orientation along the dorsal midline of the abdominal cavity, bisects the segments. (B) In the let-7-C^KO1/GKI mutant, a persistent DIOM is seen in each hemisegment (white asterisks). (A,B) In addition, let-7-C^KO1/GKI mutant DMs are smaller than wild type (arrowheads). Male-specific muscles are apparent in A5 hemisegments in both wild type and mutant (black asterisks). (C,D) Adult let-7-C^KO1/GKI mutant DMs and DM neuromuscular junctions (NMJs) do not grow to wild-type size. DMs from 2-d-old let-7-C^GKI/+; P{UAS- nls-GFP}/P{UAS-nls-GFP} (C) and let-7-C^GKI/let-7-C^KO1; P{UAS-nls-GFP}/P{UAS-nls-GFP} (D) males triple-labeled for rhodamine phalloidin (red), GFP (green), and HRP (blue). Rhodamine phalloidin-only, GFP-only, and HRP-only channels are also shown. let-7-C^KO1/GKI mutant DMs are narrower (brackets), contain fewer nuclei, and are contacted by shorter NMJs (arrowheads). (E,F) let-7-C mutant DIOMs and innervating neurons persist into adulthood. DIOMs from 0-h-old let-7-C^GKI/+; P{UAS-mCD8-GFP}/+ (E) and 12-h-old let-7-C^GKI/let-7-C^KO1; P{UAS-mCD8-GFP}/+ (F) males double-labeled for rhodamine phalloidin (purple) and GFP (green). GFP-only channels are also shown. Bars: A,B, 100 μm; C,D, 10 μm; E,F, 50 μm.