Figure 3
From: X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes
Effects of Clcn4 or Cnksr2 downregulation on morphology of mouse hippocampal neurons. Typical arborization of GFP-labeled neurons cultured for 18 days in vitro (DIV) after targeting by non-silencing (NS) or gene-specific shRNA (Clcn4 or Cnksr2) at 11 DIV. Quantification of transfected neurons, for total length of neuritic branches, total number of branches (a branch is considered as the segment between two branching points) and for dendritic branching complexity (levels were quantified per neuron from 1 to 6, each time a branching point is met from nucleus toward the distal part of each dendrite). Detection of co-transfections of shRNA and cDNA encoding plasmids for rescue experiments is shown as overlap of GFP (green) and Halotag (red) signals. Clcn4 experiment is shown in (a) and Cnksr2 in (c). More than 15 representative cells of each type were analyzed per experiment, with three independent experiments conducted. (b) Quantification of neuritic arborization in GFP expressing primary hippocampal neurons derived from Clcn4+/+ and Clcn4−/− mice as described above. Two independent experiments with>30 cells per genotype of five wild-type and four knock-out mice were analyzed. ClC-4-deficient neurons showed a significant reduction in the total number and total length of neuritic branches compared with wild-type cells. Average values with s.e.m. are shown (i) in histograms for neuritic length and number of branches and (ii) in curves for complexity levels of branching. Mann-Whitney and Chi2 tests were respectively used for statistical comparisons (ns: non-statistically significant, *P<0.05, **P<0.01, ***P<0.001). Scale bar represents 10 μm.
