Position of neocortical neurons transfected at different gestational ages with shRNA targeted against candidate dyslexia susceptibility genes

Abstract

Developmental dyslexia is a language learning disorder that affects approximately 4-10% of the population. A number of candidate dyslexia susceptibility genes have been identified, including DCDC2 and KIAA0319 on Chromosome (Chr) 6p22.2 and DYX1C1 on Chr 15q21. Embryonic knockdown of the function of homologs of these genes in rat neocortical projection cell progenitors by in utero electroporation of plasmids encoding small hairpin RNA (shRNA) revealed that all three genes disrupted neuronal migration to the neocortex. Specifically, this disruption would result in heterotopia formation (Dyx1c1 and Kiaa0319) and/or overmigration past their expected laminar location (Dyx1c1 and Dcdc2). In these experiments, neurons normally destined for the upper neocortical laminæ were transfected on embryonic day (E) 15.5, and we designed experiments to test whether these migration phenotypes were the result of targeting a specific type of projection neuron. We transfected litters with Dcdc2 shRNA, Dyx1c1 shRNA, Kiaa0319 shRNA, or fluorescent protein (as a control) at each of three gestational ages (E14.5, E15.5, or E16.5). Pups were allowed to come to term, and their brains were examined at 3 weeks of age for the position of transfected cells. We found that age of transfection did not affect the percentage of unmigrated neurons--transfection with Kiaa0319 shRNA resulted in heterotopia formation at all three ages. Overmigration of neurons transfected with Dcdc2 shRNA, while present following transfections at the later ages, did not occur following E14.5 transfections. These results are considered in light of the known functions of each of these candidate dyslexia susceptibility genes.

Figure 1. Hemispheric tracings (A,D,G), neocortical photomicrographs (B,E,H), and photomicrographs of white matter heterotopias (C,F,I) of rats embryonically transfected with either Kiaa0319 shRNA, Dcdc2 shRNA, Dyx1c1 shRNA, or fluorescent protein (Control) at either E14.5 (A,B,C), E15.5 (D,E,F), or E16.5 (G,H,I).

Bar for C,F,I = 500 µm.

Figure 2. Confocal microscopy of laminar markers CUX1 and FOXP2 following transfection of candidate dyslexia susceptibility genes at E14.5.

Small arrows indicate transfected cells co-labeled with CUX1 or FOXP2. Arrowheads indicate CUX1+ neurons that are not transfected. Large arrow for orientation with pial surface. Bar = 200 µm in all panels. A. Photomicrograph of upper lamina neurons from a rat transfected with Kiaa0319 shRNA. B. Photomicrograph of white matter heterotopia (dotted line) from a rat transfected with Kiaa0319 shRNA. C. Photomicrograph of layer 6 neurons from a rat transfected with Kiaa0319 shRNA. D. Photomicrograph of white matter heterotopia (dotted line) from a rat transfected with Kiaa0319 shRNA. E. Photomicrograph of upper lamina neurons from a rat transfected with Dcdc2 shRNA. F. Photomicrograph of white matter border (dotted line outlines corpus callosum) from a rat transfected with Dcdc2 shRNA. Str = Striatum. G. Photomicrograph of layer 6 neurons from a rat transfected with Dcdc2 shRNA. H. Photomicrograph of white matter border (dotted line) from a rat transfected with Dcdc2 shRNA. I. Photomicrograph of upper lamina neurons from a rat transfected with Dyx1c1 shRNA. J. Photomicrograph of white matter heterotopia (dotted line is white matter border) from a rat transfected with Dyx1c1 shRNA. K. Photomicrograph of layer 6 neurons from a rat transfected with Dyx1c1 shRNA. L. Photomicrograph of white matter border (dotted line) from a rat transfected with Dyx1c1 shRNA. Str = Striatum

Figure 3. Confocal microscopy of laminar markers CUX1 and FOXP2 following transfection of candidate dyslexia susceptibility genes at E16.5.

Small arrows indicate transfected cells co-labeled with CUX1 or FOXP2. Arrowheads indicate CUX1+ neurons that are not transfected. Large arrow for orientation with pial surface. Bar = 200 µm in all panels. A. Photomicrograph of upper lamina neurons from a rat transfected with Kiaa0319 shRNA. B. Photomicrograph of white matter border (dotted line) from a rat transfected with Kiaa0319 shRNA. C. Photomicrograph of white matter border (dotted line) from a rat transfected with Kiaa0319 shRNA. D. Photomicrograph of upper lamina neurons from a rat transfected with Dcdc2 shRNA. E. Photomicrograph of white matter border (dotted line) from a rat transfected with Dcdc2 shRNA. F. Photomicrograph of white matter border (dotted line) from a rat transfected with Dcdc2 shRNA. G. Photomicrograph of upper lamina neurons from a rat transfected with Dyx1c1 shRNA. H. Photomicrograph of white matter heterotopia (dotted line) from a rat transfected with Dyx1c1 shRNA. Str = Striatum. I. Photomicrograph of white matter heterotopia (dotted line) from a rat transfected with Dyx1c1 shRNA. Str = Striatum

Figure 4. Quantitative analysis of transfected neuron position.

A. Line chart showing mean laminar position of neurons transfected at E14.5. B. Histogram illustrating the mean percentage (+SEM) of transfected neurons that are unmigrated following E14.5 transfection. *Differs from control, P<.05. C. Histogram of mean (+SEM) average position of transfected neurons that migrated into the neocortex following E14.5 transfection. Horizontal lines indicate laminar boundaries. D. Line chart showing mean laminar position of neurons transfected at E15.5. E. Histogram illustrating the mean percentage (+SEM) of transfected neurons that are unmigrated following E15.5 transfection. *Differs from control, P<.05. F. Histogram of mean (+SEM) average position of transfected neurons that migrated into the neocortex following E15.5 transfection. Horizontal lines indicate laminar boundaries. *Differs from control, P<.05. G. Line chart showing mean laminar position of neurons transfected at E16.5. H. Histogram illustrating the mean percentage (+SEM) of transfected neurons that are unmigrated following E16.5 transfection. *Differs from control, P<.05. I. Histogram of mean (+SEM) average position of transfected neurons that migrated into the neocortex following E16.5 transfection. Horizontal lines indicate laminar boundaries.*Differs from control, P<.05.