Figure 7.
G3BP1 deficiency elicits mTORC1-driven neuronal phenotypes in vivo
(A) IP against TSC1 (TSC1 #3) or mock (rabbit IgG). n = 2.
(B) Zebrafish larvae injected with g3bp1 MO. dpf, days post fertilization. n = 4/day.
(C) Quantitation of Rps6-pS235/236 in (B), pooled for 2+3 dpf. Shown are data points and mean ± SEM.
(D) Dorsal and lateral view of a zebrafish larva brain. P, pallium; OT, optic tectum; H, habenula; Cb, cerebellum; OB, olfactory bulb; SP, subpallium; Th, thalamus; Tub, tuberculum; T, tegmentum; HTh, hypothalamus.
(E) IF of Rps6-pS235/236 in g3bp1 MO-injected zebrafish larvae. Nuclei, blue (DAPI); dashed white lines, white matter (WM) compartments of the pallium; arrows, Rps6-pS235/236-positive cells in the WM. Scale bar, 25 μm. n ≥ 29 larvae/condition.
(F) Quantitation of Rps6-pS235/236-positive cells in the pallium in (E). Shown are data points and mean ± SEM.
(G) Quantitation of cells in the WM in (E). Data are shown as in (F).
(H) Quantitation of Rps6-pS235/236-positive cells in the WM in (E). Data are shown as in (F).
(I) Quantitation of HuC-positive cells in g3bp1 MO zebrafish larvae (24 hpf [hours post fertilization]). Shown are data points and mean ± SEM. n ≥ 10 larvae/condition.
(J) Movement speed of single HuC-positive cells. Data are shown as in (I).
(K) Track duration of single HuC-positive cells. Data are shown as in (I). Arrow, maximum track duration.
(L) Quantitation of epileptiform events in LFP recordings from the pallia of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 34 larvae/condition.
(M) Representative LFP recordings for (L).
(N) Quantitation of epileptiform events in LFP recordings from optic tecta of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 20 larvae/condition.
(O) Representative LFP recordings for (N).
(P) Neuronal activity in pallia of Tg(HuC:GCaMP5G) zebrafish larvae injected with g3bp1 MO (4 dpf). Dashed white lines, pallium; arrows, ectopic cells with high neuronal activity in the WM; yellow/orange, high neuronal activity. Scale bar, 25 μm. n ≥ 27 larvae/condition.
(Q) Quantitation of active neuronal cells in (P). Shown are data points and mean ± SEM.
(R) Quantitation of mean neuronal activity in the subpallia of Tg(HuC:GCaMP5G) zebrafish larvae injected with g3bp1 MO (4 dpf). Shown are data points and mean ± SEM. n ≥ 15 larvae/condition.
(S) Quantitation of mean neuronal activity in the WM of Tg(HuC:GCaMP5G) zebrafish larvae injected with g3bp1 MO (4 dpf). Shown are data points and mean ± SEM. n ≥ 14 larvae/condition.
(T) Quantitation of rapamycin-mediated fold reduction in the activity of single cells in the WM of Tg(HuC:GCaMP5G) zebrafish larvae injected with g3bp1 MO (4 dpf). The number of active cells in rapamycin-treated larvae was normalized to those in untreated larvae. Shown are data points and mean ± SEM. n ≥ 14 larvae/condition.
(U) Quantitation of GABAergic cells in optic tecta of Tg(dlx5a/dlx6a-EGFP) x Tg(vglut2a:loxP-RFP-loxP-GFP) zebrafish larvae injected with g3bp1 MO (4 dpf). Shown are data points and mean ± SEM. n ≥ 34 larvae/condition.
(V) Quantitation of glutamatergic cells in optic tecta of Tg(dlx5a/dlx6a-EGFP) x Tg(vglut2a:loxP-RFP-loxP-GFP) zebrafish larvae injected with g3bp1 MO (4 dpf). Data are shown as in (U).
(W) Locomotor activity of tsc2 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 26 larvae/condition.
(X) Locomotor activity of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 36 larvae/condition.
(Y) Locomotor activity of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n = 24, untreated, n = 36 ethosuximide-treated larvae/condition.