Figure 7.
Figure 7. Impeding the interaction between Bicd1 and Fignl1 in motor neurons affects RoP-like sMN axon pathfinding as Fignl1 and Kif1bβ loss of function does. (A) Whole-mount in situ hybridization with bicd1(K4) and bicd1(K18) antisense probes on 24-hpf embryos. Images are lateral views of the embryo, anterior to the left. Insets are higher magnifications of the spinal cord. Arrowheads point at SMN expression of both zebrafish bicd1 paralogues. Bracket indicates bicd1(K18) expression in Rohon–Beard sensory neurons. Scale bars, 200 µm. (B) Schematic representation of the bicd1 constructs used in co-IP assays with Fignl1-HA and their ability to bind Fignl1. (C) Western blots showing the co-IP results summarized in panel B. Co-IPs were performed from protein extracts of COS-7 cells transfected with Dr_Fignl1-HA and the different bicd1 constructs mentioned in panel B using a GFP-trap antibody. Immunoprecipitated and coimmunoprecipitated proteins were revealed by immunoblot using anti-HA and GFP antibodies. IP, immunoprecipitation; WB, Western blot. The bicd1(713–802) domain is required to bind Fignl1. (D) Neuronal-targeted overexpression of the bicd1(713–802) domain was used in a dominant-negative approach to block endogenous interaction between Bicd1 and Fign1 in neurons. Immunolabeling of 56-hpf Tg(HuC:GAL4; UAS:GFP) and Tg(HuC:GAL4; UAS:GFP-bicd1713-802) using GFP and Zn-5 antibodies. Scale bars, 25 µm. RoP-like sMN axons overexpressing GFP-bicd1713-802 were abnormally split or defasciculated (arrows) compared with GFP-negative axons (asterisk) of the same transgenic embryos or RoP-like sMN axons expressing the GFP alone (empty arrowheads, left-hand panels). (E and F) Mean number of defasciculated/split, missing or caudally projecting rostral nerves per 10 GFP-positive and -negative hemisegments of 56-hpf Tg(HuC:GAL4; UAS:GFP) (n = 13; E) and Tg(HuC:GAL4; UAS:GFP-bicd1713-802) (n = 13; F) transgenic larvae. ***, P ≤ 0.001; ns, nonsignificant; two-way ANOVA with Bonferroni’s post test. Error bars are SEM. The same experiment was reproduced three times independently and gave rise to identical statistical differences.

Impeding the interaction between Bicd1 and Fignl1 in motor neurons affects RoP-like sMN axon pathfinding as Fignl1 and Kif1bβ loss of function does. (A) Whole-mount in situ hybridization with bicd1(K4) and bicd1(K18) antisense probes on 24-hpf embryos. Images are lateral views of the embryo, anterior to the left. Insets are higher magnifications of the spinal cord. Arrowheads point at SMN expression of both zebrafish bicd1 paralogues. Bracket indicates bicd1(K18) expression in Rohon–Beard sensory neurons. Scale bars, 200 µm. (B) Schematic representation of the bicd1 constructs used in co-IP assays with Fignl1-HA and their ability to bind Fignl1. (C) Western blots showing the co-IP results summarized in panel B. Co-IPs were performed from protein extracts of COS-7 cells transfected with Dr_Fignl1-HA and the different bicd1 constructs mentioned in panel B using a GFP-trap antibody. Immunoprecipitated and coimmunoprecipitated proteins were revealed by immunoblot using anti-HA and GFP antibodies. IP, immunoprecipitation; WB, Western blot. The bicd1(713–802) domain is required to bind Fignl1. (D) Neuronal-targeted overexpression of the bicd1(713–802) domain was used in a dominant-negative approach to block endogenous interaction between Bicd1 and Fign1 in neurons. Immunolabeling of 56-hpf Tg(HuC:GAL4; UAS:GFP) and Tg(HuC:GAL4; UAS:GFP-bicd1713-802) using GFP and Zn-5 antibodies. Scale bars, 25 µm. RoP-like sMN axons overexpressing GFP-bicd1713-802 were abnormally split or defasciculated (arrows) compared with GFP-negative axons (asterisk) of the same transgenic embryos or RoP-like sMN axons expressing the GFP alone (empty arrowheads, left-hand panels). (E and F) Mean number of defasciculated/split, missing or caudally projecting rostral nerves per 10 GFP-positive and -negative hemisegments of 56-hpf Tg(HuC:GAL4; UAS:GFP) (n = 13; E) and Tg(HuC:GAL4; UAS:GFP-bicd1713-802) (n = 13; F) transgenic larvae. ***, P ≤ 0.001; ns, nonsignificant; two-way ANOVA with Bonferroni’s post test. Error bars are SEM. The same experiment was reproduced three times independently and gave rise to identical statistical differences.

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