Postnatal analysis of the effect of embryonic knockdown and overexpression of candidate dyslexia susceptibility gene homolog Dcdc2 in the rat

Abstract

Embryonic knockdown of candidate dyslexia susceptibility gene (CDSG) homologs in cerebral cortical progenitor cells in the rat results in acute disturbances of neocortical migration. In the current report we investigated the effects of embryonic knockdown and overexpression of the homolog of DCDC2, one of the CDSGs, on the postnatal organization of the cerebral cortex. Using a within-litter design, we transfected cells in rat embryo neocortical ventricular zone around embryonic day (E) 15 with either 1) small hairpin RNA (shRNA) vectors targeting Dcdc2, 2) a DCDC2 overexpression construct, 3) Dcdc2 shRNA along with DCDC2 overexpression construct, 4) an overexpression construct composed of the C terminal domain of DCDC2, or 5) an overexpression construct composed of the DCX terminal domain of DCDC2. RNAi of Dcdc2 resulted in pockets of heterotopic neurons in the periventricular region. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Dcdc2 shRNA-transfected neurons migrated in a bimodal pattern, with approximately 7% of the neurons migrating a short distance from the ventricular zone, and another 30% migrating past their expected lamina. Rats transfected with Dcdc2 shRNA along with the DCDC2 overexpression construct rescued the periventricular heterotopia phenotype, but did not affect the percentage of transfected neurons that migrate past their expected laminar location. There were no malformations associated with any of the overexpression constructs, nor was there a significant laminar disruption of migration. These results support the claim that knockdown of Dcdc2 expression results in neuronal migration disorders similar to those seen in the brains of dyslexics.

Figure 1

In situ hybridization of Dcdc2 in embryonic rat brains. Photomontages of in situ hybridization of Dcdc2 antisense and sense probes in E15 (A,B), E17 (C,D), and E19 (E,F) rat embryos. The expression of Dcdc2 is relatively ubiquitous with a modest increase in E19 in the ventricular zone, striatum, and cortical plate. Bar in all panels = 1 mm.

Figure 2

Periventricular heterotopias (PVH) in a rat embryonically transfected with shRNA targeted against Dcdc2. A. Photomicrograph of cerebral cortex of Nissl-stained section illustrating region of PVH (arrows). This animal was embryonically transfected with Dcdc2 shRNA + GFP. B. Photomicrograph of section adjacent to Panel A immunohistochemically stained for GFP. Transfected neurons are located within the PVH. C. Photomicrograph of cerebral cortex of Nissl-stained section of a rat from the “rescue” condition. This animal was embryonically cotransfected with Dcdc2 shRNA + GFP along with a human DCDC2 protein overexpression plasmid, and shows no evidence of PVH. D. Photomicrograph of section adjacent to Panel C immunohistochemically stained for GFP. There is a solitary transfected neuron in Layer 6 (arrow), but no evidence of PVH. Bar for A–D = 500 μm. E. High power photomicrograph of PVH illustrated in panel A (arrows). F. High power photomicrograph of GFP-positive neurons in PVH. In comparison with Panel E, note that not all neurons in the PVH are transfected. Bar for E, F = 100 μm. G. High power photomicrograph of box in panel F. Note that transfected neurons in the PVH have neuronal morphology, but are misoriented. Arrow indicates direction of pial surface, arrowheads denote misoriented apical dentrites. Bar = 25 μm.

Figure 3

Confocal microscopy of the laminar specific transcription factor Cux-1 in the brain of rats embryonically transfected with shRNA targeted against Dcdc2. Top row illustrates high density of Cux-1 immunoreactive neurons in layer 2/3 as expected. Neurons transfected with Dcdc2 shRNA + GFP are co-labeled with Cux-1 (arrows). Bar = 25 μm. Middle row illustrates a single neuron in layer 5 that was transfected with Dcdc2 shRNA + GFP and is co-labeled with Cux-1. Bar = 25 μm. Bottom row is large PVH that contains numerous Cux-1 positive neurons (arrows and arrowheads), only a small subset of which are co-labeled with GFP (arrows). Bar = 50 μm.

Figure 4

Hippocampal malformations in rats embryonically transfected with shRNA targeted against Dcdc2. A. Photomicrograph of Nissl-stained section of rat embryonically transfected with Dcdc2 shRNA + GFP. There is a periventricular heterotopia (white arrow) as well as a malformation of the hippocampus (arrows). Bar = 250 μm. A′ High power photomicrograph of hippocampal malformation. Arrows are for orientation with A. Bar = 100 μm. B and B′. Section adjacent to A and A′ immunohistochemically stained for GFP. Note that only a small subset of neurons in the malformation are GFP-positive. Bar for B = 250 μm, B′ = 100 μm.

Figure 5

Patterns of neuronal migration to the cerebral cortex in each of the five experimental groups. First column contains plots of GFP-positive neurons in the cerebral cortex of rats exposed to Treatment 1 (the “experimental” condition). The second column contains plots from Treatment 2 (“control” condition). The last column contains histograms representing the percent of neurons contained within each of the deciles ranging from the white matter to the pial surface. Analysis reveals significant differences in the pattern of neuronal migration only between A and B, with there being significantly greater number of neurons in both the lower and upper deciles in the Dcdc2 shRNA group as compared to the control condition.