Loss of Tardbp does not affect spinal motor neuron axon outgrowth because of alternative splicing of Tardbpl. (A) Lateral view of wild-type, maternal zygotic tardbp-/-, and maternal zygotic tardbpl-/- embryos at 30 h postfertilization (hpf) showing the five spinal motor neuron axonal projections (labeled 1–5) anterior to the end of the yolk extension stained with znp-1 used for quantitation. Anterior to the left. (Scale bar, 25 μm.) (B) Quantitation of the length of spinal motor neuron axons measured from the exit point of the spinal cord to the growth cone in wild-type (green), maternal zygotic tardbp-/- (red), and maternal zygotic tardbpl-/- embryos (orange). Error bars indicate ±SD, n e 12 embryos per experiment. n.s., not significant, Student t test. (C) Western blot analysis comparing two independent transient Tardbp knockdown experiments with respective control injected wild-type siblings. (Upper) The Western blot with the Tardbp-specific antibody anti–TDP-43 (Novus) reveals a significant reduction of Tardbp (arrow), indicating successful knockdown in lanes 2 and 4. (Lower) Probing with a pool of N-terminal Tardbpl-specific monoclonal antibodies (anti-Tardbpl 8G1) reveals a robust up-regulation of Tardbpl_tv1 upon Tardbp knockdown (lanes 2 and 4). (D) Schematic representation of genomic exon-intron organization of tardbpl (light green boxes represent 5′ and 3′ UTR; orange boxes represents coding exons) and tardbpl_tv1 (only coding exons are shown in dark orange). Enlargement of the exon 5 splice donor site of tardbpl and the corresponding sequence in tardbpl_tv1. (E) Western blot analysis with the Tardbpl_tv1 specific monoclonal antibody Tardbpl_tv1 16C8-11 detects up-regulated Tardbpl_tv1 expression at 48 hpf and in adult brain upon loss of Tardbp compared with wild-type. The anti-Tardbpl_tv1 antibody is specific because no protein is detected in tardbpl-/-. α-Tubulin serves as a loading control. Genotypes are indicated above the respective lanes. (F) Western blot analysis with an anti-human TDP-43 antibody (Sigma) that detects zebrafish Tardbp and Tardbpl_tv1. Genotypes are indicated above the respective lanes. Note that Tardbpl_tv1 is prominently detected in tardbp-/- and runs at a slightly lower molecular weight compared with Tardbp (compare lane 3 with lane 5, labeled with an asterisk). Specificity of the antibody is demonstrated in double homozygous embryos (lane 6). α-Tubulin serves as a loading control.
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