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. 2018 Mar 5;7:e31659. doi: 10.7554/eLife.31659

Figure 5. Mosaic analysis shows a local role for endogenous Neuroligin 1 during arbor growth.

Figure 5.

(A) A schematic shows the directed insertion of the FlpStop construct into the third coding intron of the Nlg1 genomic locus by recombinase mediated cassette exchange (RMCE) with a MiMIC integration site. (B) A schematic shows the mechanism of FlpStop action for the conditional disruption of endogenous Nlg1. In the initially non-disrupting (ND) orientation the splice acceptor and stop codons are on the non-coding strand and thus do not effect gene function. Upon inversion by FLP recombinase the splice acceptor and stop codons are brought into frame on the coding strand, resulting in disrupted gene expression. The cassette is locked into this disrupting (D-lock) orientation by a FLEx switch (Schnütgen et al., 2003). In addition, inversion brings the coding sequence for tdTomato into proximity with a UAS sequence, enabling GAL4 driven expression. FlpStop lines were also generated in the initially disrupting (D) orientation, which allows rescue of Nlg1 (ND-lock clones) and ‘turns on’ tdTomato (Schematic not shown). (C) Arborisations at 80 hr APF in a wild-type control (VGlut-LexA > myr::GFP). (D) Complete induction of Nlg1 D-lock (disrupting) clones using hsFlp and a long heat-shock protocols produces arbor growth defects comparable to Nlg1 mutants. (E) Germline ND-lock rescues arbor growth to a near wild-type phenotype. (F) Schematic shows the formation of Nlg1 deficient fibres from the fusion of D-lock clonal myoblasts induced using hsFlp and a short heat shock at larval L3 stage. (G) Arbor growth (visualised with VGlut-LexA) onto regions of D-lock muscle clones (Mef2-GAL4) is disrupted, whereas arbor growth on non-clonal regions is close to wild-type. (H) Model shows the rescue of Nlg1 expression in clonal fibres formed from the fusion of ND-lock clonal myoblasts induced using hsFlp and a short heat shock at larval L3 stage. (I) Arbor growth onto ND-lock muscle clones is close to wild-type, whereas growth on non-clonal regions is disrupted. Scale bars: 100 µm (C,D,E,G,I).