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. 2018 Jun 14;7:e35518. doi: 10.7554/eLife.35518

Figure 1. Tnc is expressed in neurons and muscles and concentrates at the synaptic cleft.

(A–B) Diagram of the tnc gene and the Tnc protein domains: vWFC (orange), Pro/Thr/Ser-rich, mucin specific domains (gray), and RGD motifs (red). The antigen for the anti-Tnc antibody is marked in green. (C–E) Western blot analyses of lysates from larval brains or muscles and transiently transfected S2 cells. Tnc can be detected in control but not in tnc mutants (C) and is reduced by knockdown of tnc in neurons or muscle (E). Tnc is efficiently secreted in the S2 cell media (S) compared with the cell pellet (P). (F–J) Confocal images of NMJ4 boutons of indicated genotypes stained for Tnc (green) and HRP (magenta). Low levels of Tnc surround synaptic boutons in control but not tnc mutant NMJs (F–G). Expression of Tnc in neurons but not in the muscles induces accumulation of Tnc-positive puncta in and around the NMJ boutons. The small puncta (asterisks) appear to be extracellular, as they are still present in detergent-free staining conditions (I), whereas the large aggregates (arrows) likely correspond to intracellular secretory compartments. Scale bars: 5 μm. Genotypes: tncEP(or 82)/Df (tncEP(or 82)/Df(3R)BSC655); N > tncRNAi (BG380-Gal4/+;UAS-tncRNAi/+); M > tncRNAi (UAS-tncRNAi/G14-Gal4); N > tnc (UAS-tnc/+; elav-Gal4/+); M > tnc (UAS-tnc/BG487-Gal4).

Figure 1.

Figure 1—figure supplement 1. Tnc distribution in embryonic and larval tissues.

Figure 1—figure supplement 1.

(A–B) Confocal images of control (A) and tnc mutant (B) stage 16 embryos stained for Tnc (green) and FasII (red). Tnc signals colocalize with neuronal marker FasII in control, but are not detectable in tnc mutant embryos. (C–D) Representative images of FasII-positive longitudinal axon tracks in the CNS of stage 17 embryos. tnc mutants have no apparent fasciculation defects. (E) Quantification of relative Tnc signals at NMJ4 shown in Figure 1F–H and J. (F–I) Low magnification confocal images of muscle 4 NMJ and nerve bundles stained for Tnc (green) and HRP (magenta). Tnc signals are concentrated at NMJ and in muscle nuclei (arrowhead). When overexpressed in the neurons, Tnc accumulates along the motor neuron axon (arrow) and at synaptic terminals. Muscle expressed Tnc is detectable throughout the muscle but is not enhance at the NMJs. Relative Tnc signals at the NMJ are quantified in (E). Scale bars: 20 μm. Genotypes: tncEP/Df (tncEP/Df(3R)BSC655); N > tnc (UAS-tnc/+; elav-Gal4/+); M > tnc (UAS-tnc/BG487-Gal4).
Figure 1—figure supplement 2. Addition of an HA tag does not change the distribution of overexpressed Tnc.

Figure 1—figure supplement 2.

(A–D) Confocal images of NMJ4 boutons of indicated genotypes stained for HA (green) and HRP (magenta). Similar to Tnc, neural expression of Tnc-HA induces accumulation of HA-positive puncta inside (arrows) and outside (asterisks) the NMJ boutons. When overexpressed with strong muscle promoters, Tnc-HA is no longer detectable at the NMJs (D). Scale bars: 5 μm. Genotypes: N > tnc HA (UAS-tnc-HA/+; elav-Gal4/+); M > tnc HA (UAS-tnc-HA/+; 24B-Gal4/+).