In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

Abstract

Brain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals.

Keywords: Cerebral cortex; Ferret; In utero electroporation; Inner fiber layer; Outer radial glia; Outer subventricular zone.

Fig. 1.. GFP expression in ferrets induced by in utero electroporation performed at various time points during development.

(A) In utero electroporation was performed at E35, and GFP fluorescence was examined at P0. A dorsal view of the ferret brain is shown. GFP fluorescence was clearly visible in the cerebral cortex (arrowhead). a, anterior; p, posterior. (B) GFP-positive cells in the cerebral cortex. A coronal section is shown. GFP-positive cells were clearly visible even without immunostaining. *Lateral ventricle. (C) In utero electroporation was carried out at the indicated time points during development. Coronal sections were prepared at P0 and were stained with Hoechst 33342. Magnified images of the cerebral cortex are shown. When electroporation was performed at E31, most GFP-positive cells had already moved into the cortical plate at P0 (arrowhead). When electroporation was carried out at E40, many GFP-positive cells were found in germinal zones (arrow). CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 2 mm (A), 1 mm (B) and 200 µm (C).

Fig. 2.. Distribution of GFP-positive cells and progenitor markers.

In utero electroporation was performed at E37, and sections were prepared at E40. The sections were immunostained with anti-Sox2, anti-Pax6 and anti-Tbr2 antibodies. The cerebral cortex is shown in A. The areas within the white boxes are magnified and are shown in B. CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 200 µm (A) and 100 µm (B).

Fig. 3.. The morphology of GFP-positive cells in the OSVZ.

In utero electroporation was performed at E35, and coronal sections were prepared at P0 and were stained with Hoechst 33342. Many GFP-positive cells were distributed throughout the cortex (A). The areas within the white boxes in A are magnified and are shown in B. The areas within the white boxes in B are shown in C. Note that the morphology of GFP-positive cells was clearly visible even without immunostaining. (D,E) The sections were immunostained with anti-Sox2 antibody (D) and anti-Pax6 antibody (E), and high magnification images of the OSVZ are shown. The GFP-positive cells (arrowheads) expressed Sox2 (D) and Pax6 (E), and had basal fibers but not apical fibers, suggesting that these cell are oRG cells. GFP-positive fibers running tangentially in the inner OSVZ were also visible (arrow). *Lateral ventricle. CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 1 mm (A), 500 µm (B), 100 µm (C) and 20 µm (D,E).

Fig. 4.. Double labeling of OSVZ cells using in utero electroporation.

Cortical cells were co-transfected with pCAG-GFP and pCAG mCherry using in utero electroporation at E35. The brain was dissected out at P0, and coronal sections were made. (A) Numerous GFP-positive cells and mCherry-positive cells were distributed throughout the cortex. The areas within the white boxes, which correspond to the OSVZ, were magnified and are shown in B. Note that most GFP-positive cells in the OSVZ were also positive for mCherry. Scale bars: 1 mm (A) and 200 µm (B).

Fig. 5.. Distribution patterns of GFP-positive cells and IFL-like fibers during development.

In utero electroporation was performed at E35–37, and the distribution patterns of GFP-positive cells and IFL-like fibers in the cerebral cortex were examined at the indicated time points during development. The coronal sections of the cerebral cortex were stained with Hoechst 33342. The areas in the white boxes were magnified and are shown in B. At E40, GFP-positive cells were mainly found in germinal zones. Note that basal fibers that ascend toward the pia are clearly visible (arrowheads in A and B). GFP-positive cells were distributed throughout the cortex at P2 and were mostly found in the cortical plate at P10. GFP-positive IFL-like fibers were found preferentially in the lower OSVZ (arrows in A and square bracket in B). CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 500 µm (A, P2 and P10), 200 µm (A, E40; B, P2) and 100 µm (B, E40).

Fig. 6.. Expression of neurofilament-M in IFL-like fibers.

Sections were prepared at E40 and stained with anti-neurofilament-M antibody. Confocal images showed that GFP-positive IFL-like fibers were also positive for neurofilament-M (NF-M). Magnified images of the areas within the white boxes are shown on the right (A,B). Scale bar: 20 µm.