MZrgs4 embryos show motility and motoneurons axon outgrowth defects. (A) Quantification of the percentage of embryos that showed a normal response to the touch test at 48 hpf in WT (average of 94.44 ± 5.556, n = 18) and MZrgs4 embryos (average of 30.77 ± 7.48, n = 39). (B,C) Z projections of confocal microscopy images of HuC:gfp (B) and HuC:gfp/MZrgs4 mutant (C) showing the extending axons of primary motoneurons at 22 hpf. Arrows indicate the tip ends of extending axons. Scale bar = 20 μm. (D) Quantification of the motor axons length at 22 hpf in HuC:gfp (average of 30.20 ± 1.78 μm, 25 motor axons, n = 9) and MZrgs4/HuC:gfp (average of 31.14 ± 1.01 μm, 36 motor axons, n = 12). (E–H) Z Projections of whole-mount immunostaining for Znp1 showing the extending axons of primary motoneurons at 30 hpf in WT (E) and MZrgs4 (F) and the ramifications or branching of these motoneurons at 48 hpf in WT (G) and MZrgs4 embryos (H) Scale bar = 25 μm. (I) Quantification of the motor axons length at 30 hpf in WT (average of 106.7 ± 2.44 μm, 42 motor axons, n = 12) and MZrgs4 embryos (average of 64.37 ± 6.66 μm, 50 motor axons, n = 12). (J) Quantification of the number of ramifications per motoneuron at 48 hpf in WT (average of 20.64 ± 1.10, 36 motoneurons, n = 11) and MZrgs4 embryos (average of 9.37 ± 0.86, 53 motoneurons, n = 16). (K) Acetylated tubulin expression in WT and MZrgs4 embryo at 48 hpf showing the PLLn nerve (white arrows). Scale bar = 50 μm.