TDP-43 aggregation in zebrafish is specific. BiFC assay to determine TDP-43 aggregation. a–c Graphical illustration of the workflow. Male and female zebrafish are set up to collect the fertilized eggs (a). Eggs at the 1–2-cell stage are microinjected (b) with a combination of BiFC mRNA as illustrated below. Embryos are raised and BiFC complementation is visualized using a microscope (c). d–f Top schematics illustrate the different injection combinations. d TDP-43-aggregation and respective fluorescent signal in zebrafish somites at 24 hpf. e TDP-43-independent signal (noise). f Control (background) fluorescence at 24 hpf. Scale bar represents 20 μm. g Comparison of BiFC intensity after standard and competitive TDP-43 BiFC injections. The bar diagram shows the quantitative comparison of the normalized BiFC intensity in this fluorescence complementation assay. Dot points represent individual fish and data were pooled from 4 independent experiments. ****P < 0.0001

Optimized mVenus fragment (VN155-I152L) increases the signal-to-noise ratio of TDP-43 complementation. a Representative microscope images demonstrating the fluorescence reconstitution in muscle tissue at 24 hpf using VN155-TDP-43 versus VN155-I152L-TDP-43 constructs. Scale bars represent 20 μm. b Quantitative comparison of signal-to-noise ratio using standard VN155 versus VN155-I152L fragments. Data was obtained using an unbiased plate reader. Data pooled and averaged from three independent replicates. c, d The background fluorescence (noise) was significantly reduced with the I152L mutation (c), while the maximum fluorescence intensity did not change (d). Data in c, d was obtained using an unbiased plate reader. Data pooled from three independent replicates. *P < 0.05; ****P < 0.0001; ^nsP > 0.05

TDP-43 BiFC can be detected in muscle cells and motor neurons and is predominantly nuclear. a A representative image of a 24 hpf embryo injected with our TDP-43 BiFC constructs. Scale bar, 200 μm. b Fluorescent intensity as a percentage of signal at 24 hpf. c, d Representative pictures of the human wild-type TDP-43 BiFC signal at 24 hpf in muscle cells (c) and spinal cord motor neurons (d). e, f Representative pictures of the human wild-type TDP-43 BiFC signal at 48 hpf in muscle cells (e) and spinal cord motor neurons (f). g, h Representative pictures of the human wild-type TDP-43 BiFC signal at 72 hpf in muscle cells (g) and spinal cord motor neurons (h). Scale bar, 30 μm

Mutant TDP-43 BiFC is mislocalized to the cytoplasm. a, c, e Schemes of fluorophores used in this TDP-43 BiFC assays. b Quantification of fluorescence intensity demonstrates that mutant TDP-43 BiFC is shifted to the cytoplasm with no change to overall fluorescence intensity. d, f Representative fluorescence images of the wild-type and mutant TDP-43 BiFC signal at 36 hpf in the somites over the yolk extension. Scale bars, 10 μm

Wild-type and cytoplasmic localized mutant Fus BiFC assay in zebrafish. a, b, d, f Schemes of fluorophores used in this Fus BiFC assays. c Representative fluorescence images of the zebrafish wild-type Fus BiFC signal at 28 hpf in the somites over the yolk extension. e Mutant Fus BiFC signal is observed as discrete puncta in the cytoplasm. g The combination of mutant and wild-type Fus constructs reconstitutes primarily nuclear. Scale bars, 10 μm