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Figure 6. Knockdown of EIF3a, RPS17, and FUS blocks visual experience-dependent structural plasticity. (A, C) Schematics of different steps of regulation in gene and protein expression. We tested the effect of manipulating translation alone (A) or both translation and RNA splicing (C). (B–B’, D–D’) VE-dependent changes in growth rate over 4 hr in dark and VE in tecta treated with control MO (B, D) or designated CPP MO mixtures (B’, D’). Gray lines connect data points for individual neurons and black lines are average changes in growth rate in dark and VE. (B, B’) VE-dependent changes in growth rate in neurons from tecta electroporated with control MO (B) or eIF3A and RPS17 MO (B’). Control MO: n = 9 cells; eIF3A MO + RPS17 MO: n = 11 cells. Knocking down both eIF3A and RPS17 blocked the VE-dependent increase in dendritic arbor growth. (D, D’) VE-dependent changes in growth rate of neurons in tecta electroporated with control MO (D) or a mixture of eIF3A MO, RPS17 MO, and FUS MO (D’). Control MO: n = 7 cells; eIF3A MO + RPS17 MO + FUS MO: n = 17 cells. Combined knockdown of eIF3A, RPS17, and FUS blocked the VE-dependent increase in dendritic arbor growth. (E, F) Dendritic arbor growth rate over 4 hr in dark (E) and VE (F) in tecta electroporated with control MO or designated CPP MOs. Dendritic growth rates in dark (E) are similar to individual MO knockdown but growth rates over 4 hr in VE (F) were significantly decreased in tecta electroporated with eIF3A MO + RPS17 MO or eIF3A MO + RPS17 MO + FUS MO. The variances in growth rates in the dark were not significantly different between groups (O'Brien, Brown-Forsythe, Levene, and Bartlett test). (G) TDBL at T1 reflects developmental dendritic arbor growth. Combined knockdown of eIF3A, RPS17, and FUS or eIF3A and RPS17 significantly reduced TDBL at T1. (E–G) Control MO: n = 38 cells; eIF3A MO: n = 17 cells; FUS MO: n = 9 cells; RPS17 MO: n = 10 cells; eIF3A MO + RPS17 MO: n = 11 cells; eIF3A MO + RPS17 MO + FUS MO: n = 17 cells. (H) Percentage of cells that increased dendritic arbor growth rate in response to VE. Control MO: 97%; eIF3A MO: 59%; FUS MO: 67%; RPS17 MO: 70%; eIF3A MO + RPS17 MO: 55%; eIF3A MO + RPS17 MO + FUS MO: 29%. Triple knockdown of eIF3A, RPS17, and FUS resulted in the lowest percentage of VE-responsive cells. Control morpholino and triple knockdown conditions have significantly different proportions of cells that respond to VE compared to the rest of the groups. *p<0.05, **p<0.01, two-tailed paired Student’s t test were used to compare between two matched pairs (B, D) or Steel-Dwass test with control for nonparametric multiple comparisons (E–G). The Chi-Square test for independence with a Bonferroni correction was used to compare distributions of each group with rest of the groups (H). Error bars represent ±SEM (E–G). Image published in: Liu HH et al. (2018) © 2018, Liu et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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