The effects of embryonic knockdown of the candidate dyslexia susceptibility gene homologue Dyx1c1 on the distribution of GABAergic neurons in the cerebral cortex

Abstract

Developmental dyslexia is a language-based learning disability, and a number of candidate dyslexia susceptibility genes have been identified, including DYX1C1, KIAA0319, and DCDC2. Knockdown of function by embryonic transfection of small hairpin RNA (shRNA) of rat homologues of these genes dramatically disrupts neuronal migration to the cerebral cortex by both cell autonomous and non-cell autonomous effects. Here we sought to investigate the extent of non-cell autonomous effects following in utero disruption of the candidate dyslexia susceptibility gene homolog Dyx1c1 by assessing the effects of this disruption on GABAergic neurons. We transfected the ventricular zone of embryonic day (E) 15.5 rat pups with either Dyx1c1 shRNA, DYX1C1 expression construct, both Dyx1c1 shRNA and DYX1C1 expression construct, or a scrambled version of Dyx1c1 shRNA, and sacrificed them at postnatal day 21. The mothers of these rats were injected with BrdU at either E13.5, E15.5, or E17.5. Neurons transfected with Dyx1c1 shRNA were bi-modally distributed in the cerebral cortex with one population in heterotopic locations at the white matter border and another migrating beyond their expected location in the cerebral cortex. In contrast, there was no disruption of migration following transfection with the DYX1C1 expression construct. We found untransfected GABAergic neurons (parvalbumin, calretinin, and neuropeptide Y) in the heterotopic collections of neurons in Dyx1c1 shRNA treated animals, supporting the hypothesis of non-cell autonomous effects. In contrast, we found no evidence that the position of the GABAergic neurons that made it to the cerebral cortex was disrupted by the embryonic transfection with any of the constructs. Taken together, these results support the notion that neurons within heterotopias caused by transfection with Dyx1c1 shRNA result from both cell autonomous and non-cell autonomous effects, but there is no evidence to support non-cell autonomous disruption of neuronal position in the cerebral cortex itself.

Figure 1

Qualitative (A) and Quantitative (B) illustration of the position of transfected neurons in each of the experimental groups. A. Tracings of transfected hemispheres from each of the groups illustrating the positions of transfected neurons. B. Line graph illustrating the percent of transfected neurons throughout the depth of the cerebral cortex. There is a significant difference in the distribution of transfected neurons between the Dyx1c1 shRNA group and both the scrambled and overexpression group. This is due to more transfected neurons in the white matter and in upper cortical laminae in the Dyx1c1 shRNA transfection group (P < .05). There is no difference between the rescue and Dyx1c1 shRNA group.

Figure 2

GABAergic neurons in white matter heterotopias following in utero electroporation of Dyx1c1 shRNA. A. Low power photomicrograph of Nissl-stained section from a rat embryonically transfected with Dyx1c1-scram shRNA (control condition). There are no heterotopias visible (compare with B). B. Low power photomontage of Nissl-stained section from a rat embryonically transfected with Dyx1c1 shRNA + GFP illustrating large collection of heterotopic neurons in the white matter. White box illustrates enlarged area in panel C. Bar for A and B = 1 mm. C. High power photomontage of the Nissl-stained collection of heterotopic neurons shown in panel B. D. High-power photomontage of section adjacent to panel C immunohistochemically stained for GFP. Comparing with panel C indicates that there are large numbers of non-transfected neurons in the heterotopia. E-G. High-power photomontage of sections adjacent to panel C immunohistochemically stained for PV (E), CR (F), and NPY (G). The presence of these GABAergic interneurons in the heterotopia is supportive of non-cell autonomous effects of Dyx1c1 shRNA transfection. Bar = 250 μm.

Figure 3

Confocal microscopy illustrating E 13.5 BrdU neurons in white matter heterotopias, and co-labeling with GABAergic neurons. A–D. Heterotopic collection of neurons containing transfected neurons (A), PV+ neurons (B), E13.5 BrdU+ neurons (C). Arrows indicate PV+ neurons both within the heterotopia and in the overlying cerebral cortex. There are no transfected neurons co-labeled with either BrdU or PV. E–F. Heterotopic collection of neurons containing transfected neurons (E), CR+ neurons (F), E13.5 BrdU+ neurons (G). Arrows indicate CR+ neurons both within the heterotopia and in the overlying cerebral cortex. There are no transfected neurons co-labeled with either BrdU or CR. I–L. Heterotopic collection of neurons containing transfected neurons (I), NPY+ neurons (J), E13.5 BrdU+ neurons (K). Arrows indicate NPY+ neurons both within the heterotopia and in the overlying cerebral cortex. There are no neurons that are co-labeled with any of the antibodies. Bar = 100 μm. Long arrows for orientation toward pial surface.

Figure 4

Confocal microscopy illustrating E15.5 BrdU neurons in white matter heterotopias, and co-labeling with GABAergic neurons. A–D. Heterotopic collection of neurons containing transfected neurons (A), PV+ neurons (B), E15.5 BrdU+ neurons (C). White arrows indicate PV+ neurons, and yellow arrows indicate neurons co-labeled with PV and BrdU. Yellow arrowheads indicate transfected neurons that are co-labeled with BrdU. White arrowheads are BrdU positive neurons in the heterotopia that are not co-labeled. E–F. Heterotopic collection of neurons containing transfected neurons (E), CR+ neurons (F), E15.5 BrdU+ neurons (G). White arrows denote CR+ neurons, and yellow arrows indicate neurons co-labeled with CR and BrdU. Yellow arrowheads indicate transfected neurons that are co-labeled with BrdU. White arrowheads are BrdU positive neurons in the heterotopia that are not co-labeled. I–L. Heterotopic collection of neurons containing transfected neurons (I), NPY+ neurons (J), E15.5 BrdU+ neurons (K). White arrows denote NPY+ neurons, and yellow arrows indicate a neuron co-labeled with NPY and BrdU. Yellow arrowheads indicate transfected neurons that are co-labeled with BrdU. White arrowheads are BrdU positive neurons in the heterotopia that are not co-labeled. Bar = 100 μm. Long arrows for orientation toward pial surface.

Figure 5

Confocal microscopy illustrating E 17.5 BrdU neurons in white matter heterotopias, and co-labeling with GABAergic neurons. A–D. Heterotopic collection of neurons containing transfected neurons (A), PV+ neurons (B), E17.5 BrdU+ neurons (C). White arrows indicate PV+ neurons, and the yellow arrow indicates a neuron co-labeled with PV and BrdU. Arrowheads are BrdU positive neurons in the heterotopia that are not co-labeled. There are no transfected neurons co-labeled with either BrdU or PV. E–F. Heterotopic collection of neurons containing transfected neurons (E), CR+ neurons (F), E17.5 BrdU+ neurons (G). White arrows indicate CR+ neurons, and yellow arrows indicate neurons co-labeled with CR and BrdU. Arrowheads are BrdU positive neurons in the heterotopia that are not co-labeled. There are no transfected neurons co-labeled with either BrdU or CR. I–L. Heterotopic collection of neurons containing transfected neurons (I), NPY+ neurons (J), E17.5 BrdU+ neurons (K). White arrows indicate NPY+ neurons. Arrowheads are BrdU positive neurons in the heterotopia that are not co-labeled. There are no neurons that are co-labeled with any of the antibodies in the heterotopia. Bar = 100 μm. Long arrows for orientation toward pial surface. wm = white matter.

Figure 6

Line graphs illustrating the percent of PV+, CR+, and NPY+ neurons throughout the cortical depth for all four transfection groups. A. There are significantly greater numbers of PV+ neurons in white matter in the Dyx1c1 shRNA-transfected group compared to the scrambled and expression groups, respectively (P< .05). There is no evidence of disruption of neuronal position of PV+ in the cerebral cortex. B. There is no evidence of disruption of neuronal position of CR+ in the cerebral cortex. C. There is no evidence of disruption of neuronal position of NPY+ in the cerebral cortex, although there are significantly more NPY+ neurons in the white matter in the Dyx1c1 shRNA + DYX1C1 Expression condition (P < .05).