Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus

Abstract

To investigate the role of Dicer and microRNAs in the mammalian CNS, we used mice in which the second RNase III domain of Dicer was conditionally floxed. Conditional Dicer mice were bred with mice expressing an alpha-calmodulin kinase II Cre to selectively inactivate Dicer in excitatory forebrain neurons in vivo. Inactivation of Dicer results in an array of phenotypes including microcephaly, reduced dendritic branch elaboration, and large increases in dendritic spine length with no concomitant change in spine density. Microcephaly is likely caused by a 5.5-fold increase in early postnatal apoptosis in these animals as determined by active caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) staining in the cortex. Loss of Dicer function had no measurable effect on cortical lamination as determined by in situ hybridization, suggesting that microcephaly is not caused by defects in neuronal migration. Together, these results illustrate the in vivo significance of Dicer and miRNAs in the mammalian CNS and provide additional support for previous in vitro studies indicating that misregulation of this pathway may result in gross abnormalities in cell number and function that may contribute to a variety of neurological disorders.

Figure 1.

Cre recombination occurs throughout the forebrain and results in early death. A, Dicer targeting construct. The second RNase III domain of Dicer was flanked with Lox P sites. After Cre recombination at embryonic day 15.5, Dicer is rendered inactive, leading to a failure to produce microRNAs in excitatory forebrain postmitotic neurons. B, Kaplan–Meier plot of survival demonstrates that 40% of Dicer^flox/flox; CaMKII Cre (n = 26; open triangle) animals die by postnatal day 2 and that the remaining animals die by the end of postnatal day 21, whereas other genotypes persist into adulthood (Dicer^flox/flox, n = 20, open square; Dicer^flox/wt, n = 20, open circle; Dicer^flox/wt; CaMKII Cre, n = 20, closed square). C–H, Floxed Dicer mice were crossed to α-CaMKII Cre- and RosaR26R-expressing mice to visualize brain regions in which Dicer was removed. C, Cre-recombined cells at postnatal day 21 in a Dicer^flox/wt; CaMKII Cre; R26R ^+/− mouse. Recombination is high throughout all cortical layers as well as throughout the pyramidal cell layer of the hippocampus. Little expression/recombination is observed in the thalamus. D, Cre recombination at postnatal day 21 in a Dicer^flox/wt; CaMKII Cre; R26R ^+/+ mouse demonstrating the specificity of X-gal staining. E, X-gal staining at the level of the cerebellum in a Dicer^flox/wt; CaMKII Cre; R26R ^+/− mouse illustrating forebrain specificity of the CaMKII Cre line. F, Similar to the image in D, there is a lack of X-gal staining in the cerebellum in Dicer^flox/wt; CaMKII Cre; R26R ^+/+ mice. G, Higher magnification of cortical layers IV–VI. Recombination occurs in most but not all cortical neurons. H, Higher magnification of the hippocampal formation in a Dicer^flox/wt; CaMKII Cre mouse illustrating that recombination occurs throughout the dentate gyrus and CA1–3 regions, but not in strata, where interneurons and glia predominate. Scale bars: C–F, 1 mm. RBD, RNA binding domain; FRT, flipase recognition target.

Figure 2.

MicroRNA levels are decreased in Dicer mutant mice. A, Northern blot analysis for mir-132 at P15 demonstrates a decrease in miRNA levels in the cortex and hippocampus of Dicer^flox/flox; CaMKII Cre mice. B, qRT-PCR analysis of mir-132 levels in the cortex and hippocampus of Dicer mutant mice at P21 demonstrates that miRNA levels continue to decrease over time. U6 was used as a loading control in all experiments. Five micrograms of total RNA were loaded per well for cortical and hippocampal mir-132 Northern blot analysis. Twenty nanograms of total RNA were used to generate cDNA for qRT-PCR analysis. Error bars represent the SEM. Values are statistically significant using a one-way ANOVA (*p < 0.001) with a Tukey's multiple comparison-post test.

Figure 3.

Dicer mutant animals show decreases in brain weight and abnormally large ventricles. A, Brains of Dicer mutant animals are consistently smaller than their wild-type littermates. The arrow highlights increased exposure of superior colliculus in Dicer mutant animals. B, Quantification of brain weight (N indicates number of animals). C, D, Dicer^flox/flox (C) and Dicer^flox/flox; CaMKII Cre ^+/− (D) mice immunostained for neuropilin-2 demonstrate substantial differences in ventricular size as well as axonal pathfinding abnormalities (arrows in C and D). Arrowheads illustrate differences in anterior commissure. Scale bars: A, 1 mm; C, D, 500 μm. In B, error bars represent SEM. Values are statistically significant (*p < 0.001) using a one-way ANOVA with a Tukey's multiple comparison post test.

Figure 4.

Dicer inactivation leads to increased cortical apoptosis in vivo. A, Quantification of active caspase-3 staining in Dicer^flox/wt; CaMKII ^+/− and Dicer^flox/flox; CaMKII ^+/− at postnatal day 0 (Dicer^flox/flox; CaMKII Cre ^+/−, n = 25, N = 3; Dicer^flox/wt; CaMKII Cre ^+/−, n = 24, N = 3). For negative controls, n = 12 and N = 3. N indicates the number of animals, and n the number of sections analyzed. B, Immunostaining for the active (cleaved) form of caspase-3 illustrating neurons adjacent to the lateral ventricle undergoing programmed cell death. Inset, Apoptotic layer 5 cortical neuron (same section) showing blebbing characteristic of programmed cell death. C, TUNEL staining of Dicer^flox/flox (top) and Dicer^flox/flox; CaMKII Cre animals (bottom) at postnatal day 0 indicating that Dicer mutant animals have multifold increases in cortical apoptosis with the majority of apoptosis occurring at or around the lateral ventricles in the cortex of Dicer mutant animals. Positive controls were first treated with 1 μg/μl DNase I in 1× TBS-Tween 20/MgSO₄ for 20 min at room temperature to cause DNA damage and then processed with other sections. In A and C, negative controls refer to samples untreated with an anti-caspase-3 antibody or terminal deoxynucleotidyl transferase, respectively. Scale bars: B, 10 μm; C, 100 μm. In A, error bars represent SEM. Values are statistically significant using a one-way ANOVA (*p < 0.001) with a Tukey's multiple comparison post test.

Figure 5.

Dendritic branching in hippocampal neurons is impaired in Dicer mutant animals. A, Quantification of basal and apical dendritic branch points in Dicer^flox/flox and Dicer^flox/flox; CaMKII ^+/− Cre animals demonstrate decreases in the number of dendritic branch points. B, Example of Golgi-stained hippocampal CA1 neurons from Dicer^flox/flox and Dicer^flox/flox; CaMKII ^+/− animals illustrating differences in basal and apical branching. C, Sholl analysis of Dicer^flox/flox and Dicer^flox/flox; CaMKII ^+/− animals illustrates substantial differences in dendritic complexity (Dicer^flox/flox, n = 24, N = 3; Dicer^flox/flox; CaMKII ^+/−, n = 24, N = 3). D, Golgi-stained Dicer^flox/flox hippocampus with stereotyped architecture including a well defined dentate gyrus and CA1–3 region. E, Golgi-stained Dicer^flox/flox; CaMKII ^+/− Cre hippocampus outlined in white illustrates decreased size relative to that shown in E (images taken at same magnification). Abnormal hippocampal architecture observed in Dicer^flox/flox; CaMKII ^+/− Cre mice makes distinct layers of the CA region difficult to discern. The white arrow highlights loss of the dentate gyrus. Images were taken at 4× in D and E. n is the number of neurons, N the number of animals. Scale bars: B, 10 μm; E, F, 100 μm. Error bars represent SEM. Values in A are statistically significant using a one-way ANOVA (*p < 0.001) with a Tukey's multiple comparison post test. Values in C are significant using an unpaired two-tailed t test (*p < 0.05).

Figure 6.

Inactivation of Dicer in hippocampal CA1 neurons leads to increased apical dendritic spine length but not density in vivo. A, Spontaneous action potentials and synaptic activity from Dicer mutant animals at postnatal day 15 indicate that dendritic spines are functional. B, Mean dendritic spine length for Dicer^flox/flox, 0.98 ± 0.04 (n = 245, N = 2); Dicer^flox/flox; CaMKII ^+/−, 2.40 ± 0.23 (n = 117, N = 2); and Dicer^flox/wt, 0.94 ± 0.03 (n = 236, N = 3); Dicer^flox/wt; CaMKII ^+/−, 1.26 ± 0.08 (n = 141, N = 3). C, Example of dendritic spines in Dicer^flox/flox; CaMKII ^+/− Cre ^−/−, and Dicer^flox/wt; CaMKII ^+/− Cre ^+/− animals. Dendritic spines of Dicer^flox/flox; CaMKII ^+/− Cre (as shown in C, left) were observed up to 12 μm in length. D, Cumulative distribution of apical dendritic spine length in each Dicer genotype. Loss of microRNAs results in one-third of dendritic spines being greater in length than the longest measured dendritic spine in other Dicer genotypes. E, F, Basal and apical dendritic spine density in CA1 hippocampal neurons at postnatal day 21 in Golgi-stained neurons. The number of spines per 50 μm dendrite were counted by three blind observers, and the numbers were averaged. Criterion for inclusion included dendrites of 75 μm or longer. For analysis, spines were counted on basal or apical dendrites starting 25 μm from the cell body for a length of 50 μm. Analysis performed on two to three dendrites per cell in each condition with four to five cells measured. Three to four animals were used per condition. N is the number of animals, and n the number of cells. Scale bars, 2 μm. Error bars represent SEM. Values in B are statistically significant (*p < 0.001) using a one-way ANOVA with a Tukey's post test.