Characterization of rgs4 mutant. (A) Schematic representation of the rgs4 genomic locus. The extended region on the exon 2 represents the sequence targeted by the CRISPR/Cas9 system. Red: sgRNA binding site. Blue: PAM sequence. Rgs4⁺ corresponds to the wild-type allele; rgs4⁻ is the loss-of-function allele used in this study. Dashes represent the 2 base pairs deletion. (B) Schematic of the wild type Rgs4 protein (Rgs4⁺) and the mutated Rgs4 protein (Rgs4⁻). The 215 amino acid (aa) long Rgs4⁺ protein contains the RGS domain from amino acid 71 to 183. In rgs4^−/− mutant fish, the 2 base pairs deletion results in a frame shift from the amino acid 28 generating a premature STOP codon at the level of the amino acid 64. (C) Lateral views of WT and MZrgs4 embryos at 48 hpf; black arrow designates a slight heart oedema observed in MZrgs4 embryos. Scale bar = 200 µm.

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.

Rgs4 is required for neuronal development in the PLLg and spinal cord. (A–D), single planes of confocal microscopy images representing the PLLg following HuC labelling at 48 and 72 hpf in WT (A,C) and MZrgs4 (B,D). Scale bars = 5 μm. (E) Graph showing the number of neurons within the PLLg in WT (average of 56.70 ± 1.43, n = 10 at 48 hpf; average of 71.17 ± 1.25, n = 12 at 72 hpf) and MZrgs4 embryos (average of 29.14 ± 1.71, n = 14 at 48 hpf; average of 52.14 ± 1.33, n = 21 at 72 hpf) at 48 and 72 hpf. (F) Quantification of the number of neurons in the PLLg following rgs4 mRNA injection in MZrgs4 mutants at 48 hpf (average of 54.89 ± 1.01 neurons in WT, n = 9; average of 28.65 ± 1.43 neurons in MZrgs4, n = 17; average of 53.15 ± 1.13 neurons in MZrgs4 + rgs4mRNA, n = 13). (G,H) single planes of confocal microscopy images of Tg(HuC:gfp) (G) and Tg(HuC:gfp)/MZrgs4 (H) at 48 hpf. Scale bar = 20 μm. (I) Quantification of the number of HuC:gfp + cells within the same area of the spinal cord in WT (average of 129 ± 2.70 gfp + cells, n = 12) and MZrgs4 (average of 110.7 ± 2.07 gfp + cells, n = 12) at 48 hpf.

MZrgs4 embryos show a specific decrease in the number of HuC+ cells and a significant increase in the number of PH3+ cells in the spinal cord. (A) Lateral views of spinal cord Z projections (around 15 μm) following whole-mount immunostaining for HuC (left) and counterstained with DAPI (middle) with merged still images (right) in WT (top) and MZrgs4 larvae (bottom) at 48 hpf. Scale bar = 20 μm. (B) Quantification of the number of HuC+ and HuC− cells within the spinal cord in WT (average of 101.2 ± 3.12 for HuC+ ; average of 93.83 ± 7.36 for HuC−, n = 6) and MZrgs4 larvae (average of 51.86 ± 5.25 for HuC+ ; average of 84.71 ± 4.39 for HuC−, n = 7) at 35 hpf (left) and at 48 hpf in WT (right) (average of 166.7 ± 7.04 for HuC+ ; average of 146.2 ± 8.28 for HuC−, n = 6) and MZrgs4 larvae (average of 130.3 ± 3.7 for HuC+ ; average of 153.1 ± 3.99 for HuC−, n = 7). (C) Lateral views of spinal cord Z projections (around 30 μm for PH3) following whole-mount immunostaining for PH3 (left) and HuC (middle) with merged still images (right) in WT (top) and MZrgs4 larvae (bottom) at 48 hpf. (D) Quantification of the number of PH3+ cells within the spinal cord in WT (average of 14.29 ± 1.82 at 35 hpf, n = 7; average of 4 ± 1.08 at 48 hpf, n = 9) and MZrgs4 larvae (average of 19.56 ± 1.10 at 35 hpf, n = 9; average of 12.44 ± 1.51 at 48 hpf, n = 9). (E) Dorsal views of head Z projections (around 400 μm) following HuC whole-mount immunostaining in WT and MZrgs4 larvae at 48 hpf. Dashed lines represent the nervous system. Scale bar = 50 μm. (F) Quantification of the head area in WT (average of 180,194 ± 3289, n = 9) and MZrgs4 larvae (average of 153,316 ± 5151, n = 9) at 48 hpf. (G) Quantification of the head perimeter in WT (average of 1677 ± 31.90, n = 9) and MZrgs4 larvae (average of 1564 ± 30.24, n = 9) at 48 hpf.

Loss of rgs4 function alters Akt signaling in neural cells. (A,B) Immunoblotting of lysates from zebrafish embryos at 20 hpf. Akt and p-Akt amounts were normalized to α-tubulin and the ratio of p-Akt relative to Akt was compared between WT and MZrgs4 lysates. No significant difference was observed between the two groups. Erk and p-Erk amounts were normalized to α-tubulin and the ratio of p-Erk relative to Erk was compared between WT and MZrgs4 lysates. No significant difference was observed between the two groups. (C) Immunoblotting of lysates from zebrafish embryos at 48 hpf. Akt and p-Akt amounts were normalized to β-actin and the ratio of p-Akt relative to Akt was compared between WT and MZrgs4 lysates. MZrgs4 embryos showed a significant decrease in the amount of p-Akt/Akt in comparison to WT. (D) Immunoblotting of lysates from zebrafish embryos at 48 hpf. Erk and p-Erk amounts were normalized to Gapdh and the ratio of p-Erk relative to Erk was compared between WT and MZrgs4 lysates. No significant difference was observed between the two groups. (E) Immunoblotting of neural lysates (or extracts) from zebrafish embryos at 48 hpf. Akt and p-Akt amounts were normalized to β-actin and the ratio of p-Akt relative to Akt was compared between WT and MZrgs4 lysates. MZrgs4 embryos showed a significant decrease in the amount of p-Akt/Akt in comparison to WT.

Akt and Erk activities mediate Rgs4-dependent PLLg development and motoneurons branching. (A) Quantification of the number of neurons in the PLLg in WT (average of 55.83 ± 1.02, n = 18), WT + caAkt (average of 51.18 ± 0.65, n = 11), WT + caErk (average of 49.90 ± 1.35, n = 10), MZrgs4 (average of 28.35 ± 1.28, n = 20), MZrgs4 + caAkt (average of 55 ± 1.36, n = 22) and MZrgs4 + caErk (average of 45.08 ± 0.64, n = 12). (B–G) Z projections of whole-mount immunostaining for Znp1 labeling the ramifications of motoneurons in WT (B), MZrgs4 (C) WT + caAkt (D), MZrgs4 + caAkt (E), WT + caErk (F) and MZrgs4 + caErk (G) at 48 hpf. Scale bar = 20 μm. (H) Quantification of the number of ramifications per motoneuron in WT (average of 21.08 ± 1.10, 40 motoneurons, n = 12), WT + caAkt (average of 15.10 ± 0.87, 30 motoneurons, n = 10), WT + caErk (average of 14.27 ± 0.83, 33 motoneurons, n = 11), MZrgs4 (average of 9.28 ± 0.66, 46 motoneurons, n = 14), MZrgs4 + caAkt (average of 20.20 ± 1.28, 32 motoneurons, n = 10) and MZrgs4 + caErk (average of 19.83 ± 1.00, 40 motoneurons, n = 12) at 48 hpf.

Loss of rgs4 function alters mTOR signaling in neural cells (A) Immunoblotting of lysates from zebrafish embryos at 20 hpf. S6 and p-S6 amounts were normalized to α-tubulin and the ratio of p-S6 relative to S6 was compared between WT and MZrgs4 lysates. No significant difference was observed between the two groups. Immunoblotting of lysates from zebrafish embryos at 48 hpf. S6 and p-S6 amounts were normalized to β-actin and the ratio of p-S6 relative to S6 was compared between WT and MZrgs4 lysates. MZrgs4 embryos show a significant decrease in the amount of p-S6/S6 in comparison to WT. (B) Immunoblotting of neural lysates from zebrafish embryos at 48 hpf. S6 and p-S6 amounts were normalized to β-actin and the ratio of p-S6 relative to S6 was compared between WT and MZrgs4 lysates. MZrgs4 embryos showed a significant decrease in the amount of p-S6/S6 in comparison to WT.

Rgs4 function, Akt and Erk activities that mediate Rgs4-dependent motoneurons outgrowth are all required within motoneurons. (A) Representative maximum projections of z-stack optical sections of CaP and MiP at 30 hpf following injection of different constructs allowing the expression of mcherry in WT and MZrgs4, Rgs4-P2A-mcherry in MZrgs4, caAkt-P2A-mcherry in MZrgs4 and caErk-P2A-mcherry in MZrgs4 specifically in motoneurons under the control of hb9 promoter. (B) Quantification of individual CaP motor axons length following injection of hb9:mcherry in WT (average of 84.33 ± 3.48 μm, 6 CaP, n = 4) and MZrgs4 (30.57 ± 4.41 μm, 7 CaP, n = 5), injection of hb9:rgs4-P2A-mcherry in MZrgs4 (average of 76 ± 4.91 μm, 4 CaP, n = 4), injection of hb9:caAkt-P2A-mcherry in MZrgs4 (average of 89.67 ± 8.24 μm, 6 CaP, n = 6) and injection of hb9:caErk-P2A-mcherry in MZrgs4 (average of 87.67 ± 5.73 μm, 6 CaP, n = 6). (C) Quantification of individual MiP motor axons length following injection of hb9:mcherry in WT (average of 54.20 ± 0.66 μm, 5 MiP, n = 4) and MZrgs4 (20 ± 3.23 μm, 6 MiP, n = 5), injection of hb9:rgs4-P2A-mcherry in MZrgs4 (average of 52 ± 0.82 μm, 4 MiP, n = 4), injection of hb9:caAkt-P2A-mcherry in MZrgs4 (average of 58 ± 1.92 μm, 5 MiP, n = 5) and injection of hb9:caErk-P2A-mcherry in MZrgs4 (average of 55.71 ± 2.05 μm, 7 MiP, n = 7). ns, non significant.