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. Author manuscript; available in PMC: 2023 Aug 1.
Published in final edited form as: Mol Neurobiol. 2022 Jun 7;59(8):5104–5120. doi: 10.1007/s12035-022-02905-4

Figure 2. WDR5-suppressed neurons exhibit a defect in neuronal migration to the cortical Plate.

Figure 2.

(A) shRNA-mediated WDR5-supression induced abnormal localization of electroporated cells in the brain. E14.5 mouse brains were electroporated in utero with nonsilencing shRNA (control) or shWDR5 construct. shRNAs encoded GFP in a separate reading frame for a labeling purpose. The brain samples were collected at E19.5 stages. GFP-positive or RFP-positive cells were visualized in the lateral cerebral cortex. Scale bar, 50μm. (B) Quantification of neuron positions throughout the cerebral cortex. n=5 mice for each condition. Statistical significance was determined by two-way ANOVA with Bonferonni correction test. Data shown are mean (+/−) SEM. Stars indicate significant difference when compared with controls. *p < 0.05, ***p < 0.001. (C) Illustration showing the silent mutations in the shWDR5-resistant construct of WDR5. The twelve silent mutations (red) in the shWDR5-target sequence (blue) of the WT WDR5 amino acids (black), which confer resistance to the shWDR5 and no change in the amino acid sequence compared with the WT WDR5. (D) Western blot showing insensitivity of rWDR5 versus sensitivity of WT WDR5 to shWDR5. The proteins were exogenously expressed in HEK 293T cells. n=3 independent experiments for WDR5 expression in HEK 293T cells. (E) Co-expression of rWDR5 construct restored the inhibitory effect of shWDR5 in pyramidal neural migration. Control shRNA or shWDR5 or shWDR5 with rWDR5 plasmid were electroporated in utero into E14.5 mouse embryos and observed neural migration and multipolar to bipolar transition of pyramidal neurons at E17.5. Scale bar, 50μm, 25μm. (F) Quantification of neuron positions throughout the cerebral cortex. n=5 mice for each condition. Statistical significance was determined by two-way ANOVA with Bonferonni correction test. Data shown are mean (+/−) SEM. Stars indicate significant difference when compared with controls. ***p < 0.001. (G) Co-expression of rWDR5 construct rescued the inhibitory effect of shWDR5 in multipolar to bipolar transition. Quantification of numbers either multipolar or bipolar neurons in IZ. n=5 mice for each condition. Statistical significance was determined by two-way ANOVA with Bonferonni correction test.