Journal: Journal of Virology
Article Title: Human Cytomegalovirus IE2 Protein Disturbs Brain Development by the Dysregulation of Neural Stem Cell Maintenance and the Polarization of Migrating Neurons
Figure Lengend Snippet: HCMV IE2 expression disturbs cortical brain development in vivo . (A) Schematic of gene delivery into the embryonic brain through in utero electroporation of plasmid DNA and assessment of the neurodevelopment-modulating activity of a gene of interest by determining the transgene-expressing cell positions in the cortical layers. Initially, only neural stem cells lining the VZ are electroporated, and as development proceeds, they give rise to neural stem cells that reside in the VZ or neurons that migrate up to the pial surface. (B) IE expression in the brains at 2 days postelectroporation was confirmed by immunostaining using anti-GFP (reporter gene; green) and anti-IE1/2 (red) primary antibodies as well as Alexa Fluor 488- and 555-conjugated secondary antibodies. IE2-expressing cells in E15.5 (C) or E18.5 (H and I) embryonic brains that were electroporated at E13.5 were immunolabeled using anti-GFP primary and Alexa 488-conjugated secondary antibodies. For panel C, GFP immunofluorescence merged with DAPI-counterstained images are shown on the right. (D) Quantification of GFP + cell positions from panel C. (E) Double immunolabeling of E15.5 brain sections electroporated with IE2-expressing plasmid at E13.5 using anti-GFP (green) and anti-Sox2 (red) primary antibodies. (F) The shapes of GFP + electroporated cells in E15.5 cortices at the transition between MMZ and RMZ. The dotted red lines indicate the direction of radial migration. Radial cells were defined as cells with a leading process with a deviation angle of less than 45° relative to the normal radial migration direction. (G) Quantification of cell shapes shown in panel F. (I) Examination of callosal axon trajectory (closed arrowheads) using immunolabeling of GFP at E18.5. The dotted lines indicate the midline, and an open arrowhead indicates where IE2 + callosal axons stop projection. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MMZ, multipolar morphology zone; RMZ, radial morphology zone; CC, corpus callosum; LV, lateral ventricle. Scale bars were 20 μm (B, E, and F), 50 μm (C), and 100 μm (H and I). Error bars represent SD. Student's t test was used to determine statistical significance. **, P
Article Snippet: For in utero electroporation, 1 to 2 μl of DNA solution (2 μg/μl) in PBS with 0.01% fast green dye (Sigma) was injected into the lateral ventricle of E13.5 embryos, and five square electric pulses (33 V) were delivered at one pulse per second (50-ms pulse followed by 950-ms gap) to the embryos through the uterus with forceps-type electrodes (CUY650P5; 5-mm-diameter platinum round plates; Nepagene).
Techniques: Expressing, In Vivo, In Utero, Electroporation, Plasmid Preparation, Activity Assay, Immunostaining, Immunolabeling, Immunofluorescence, Migration