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. 2007 Sep 17:7:103.
doi: 10.1186/1471-213X-7-103.

Gene delivery into mouse retinal ganglion cells by in utero electroporation

Cristina Garcia-Frigola  1 , Maria Isabel Carreres, Celia Vegar, Eloisa Herrera
Affiliations

Gene delivery into mouse retinal ganglion cells by in utero electroporation

Cristina Garcia-Frigola et al. BMC Dev Biol. .

Abstract

Background: The neural retina is a highly structured tissue of the central nervous system that is formed by seven different cell types that are arranged in layers. Despite much effort, the genetic mechanisms that underlie retinal development are still poorly understood. In recent years, large-scale genomic analyses have identified candidate genes that may play a role in retinal neurogenesis, axon guidance and other key processes during the development of the visual system. Thus, new and rapid techniques are now required to carry out high-throughput analyses of all these candidate genes in mammals. Gene delivery techniques have been described to express exogenous proteins in the retina of newborn mice but these approaches do not efficiently introduce genes into the only retinal cell type that transmits visual information to the brain, the retinal ganglion cells (RGCs).

Results: Here we show that RGCs can be targeted for gene expression by in utero electroporation of the eye of mouse embryos. Accordingly, using this technique we have monitored the morphology of electroporated RGCs expressing reporter genes at different developmental stages, as well as their projection to higher visual targets.

Conclusion: Our method to deliver ectopic genes into mouse embryonic retinas enables us to follow the course of the entire retinofugal pathway by visualizing RGC bodies and axons. Thus, this technique will permit to perform functional studies in vivo focusing on neurogenesis, axon guidance, axon projection patterning or neural connectivity in mammals.

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Figures

Figure 1
Figure 1
Gene targeting into the embryonic retina. (A) Schematic representation of retinal electroporation in utero. A small amount of DNA is injected into the embryo's eye through the uterine wall (left), and then electric pulses are passed using paddle electrodes. The result is the delivery of DNA to a subset of retinal cells (right). Only when the positive electrode was located on the injected eye was the electroporation successful. (B) Retinal section of an E16 embryo electroporated at E13. GFP expressing cells can be detected in the central part of the retina (arrows), surrounding the optic disc; scale bar: 200 μm. (C, D, E) Flattened whole mounts of E16 retinas electroporated at E13 after injection of different volumes of GFP-plasmid solution (0.2 μl, 0.5 μl and 1 μl respectively of a 1 μg/μl DNA solution) show the increase in the number cells targeted in the central retina (cells in the dashed circle). Scale bar: 500 μm.
Figure 2
Figure 2
Visualization of the development of the retinas targeted at E13. (A-I) Retinas from E13 embryos were electroporated with GFP-bearing plasmids and sacrificed at E14, E16 or E18. Left panels show retinal sections from electroporated embryos incubated with anti-Islet1/2 antibodies to detect post-mitotic RGCs. Middle panels show targeted cells in the same retinal sections. Note that axons projecting to the inner layer can already be visualized in panel B at E14. Right panels show the merged images. At E16 GFP-positive cells are located closer to the inner layer (labelled by Islet 1/2, red) and a few double-labelled cells are observed (white arrows). At E18 the majority of the electroporated cells are located in the inner retinal layer and many of them are positive for Islet1/2. Scale bar: 20 μm. (J) Diagram showing the retrograde labelling paradigm. Dextran-rhodamine is applied at E17 in the optic tract (red) contralateral to the retina that was electroporated at E13 (green). The typical distribution of dextran-labelled cells and axons in the contralateral retina at E17 are shown (red), together with the GFP targeted cells that were electroporated at E13. (K) Retinal section electroporated at E13 (green cells) and retrogradely labelled with dextran-rhodamine (red cells). In all of the merged images, the double/labelled cells are yellow and they are indicated by white arrows. Scale bar: 100 μm (L-N) High magnification of the boxed area in K. Scale bar: 25 μm INL, Inner layer; VZ, ventricular zone.
Figure 5
Figure 5
The entire retinofugal pathway can be visualized when RGCs are targeted. (A) GFP-expressing axons exiting the retina through the optic disc. (B) 24 h after electroporation many growth cones from targeted RGCs are observed at the optic chiasm. (C) Retinal axons are seen in the optic tract three days after electroporation. (D) In newborn animals electroporated at E13, individual retinal axons expressing GFP project into the superior colliculus. (E) Higher magnification of (D) showing individual axons within the superior colliculus. (F) The location of the axons from targeted cells can be detected in the LGN of frontal brain sections of P8 animals after electroporation at E13. (G) Higher magnification of (F) shows the precise location of individual axons. (H) RGC axons electroporated at E13 in the retina terminate in the superior colliculus at P8 (arrow). (I) A frontal section through the superior colliculus of the same animal shown in (H). Od, optic disc; on, optic nerve; md, midline; ot, optic tract; sc, superior colliculus; dLGN, dorsal lateral geniculate nucleus; vLGN, ventral lateral geniculate nucleus; ic, inferior colliculus; cb, cerebellum. Scale bars: 100 μm in E; 200 μm in A, B, C, F, G, I and 500 μm in D, H.
Figure 3
Figure 3
Visualization of postnatal retinas electroporated at E13. Retinas from E13 embryos were electroporated with GFP-bearing plasmids and sacrificed at P0 or P8. Retinal sections from electroporated embryos were incubated with anti-Calbindin (A-D) or anti-Brn3a (E-L) antibodies to identify horizontal cells and post-mitotic RGCs, respectively. (A-C) Calbindin staining on electroporated retinal sections at P0. (A) Shows the electroporated cell population at P0. Note that the vast majority of electroporated cells are distributed between the RGC and INL retinal layers but also, infrequent GFP labelled cells can be observed in the VZ. (B) Calbindin staining performed on electroporated retinal sections (C) Co-localization of calbindin and GFP (yellow cells) in a single cell located deep in the ventricular zone. A few amacrine cells are also positive for calbindin in the INL. Scale bar: 50 μm. (D) Higher magnification of a single cell in the ventricular zone that was electroporated at E13 and stained for calbindin at P0 indicating that it is a horizontal cell. Scale bar: 25 μm. (E-G) Sections of P0 retinas that were electroporated at E13, and stained for Brn3a. Scale bar: 50 μm. (I-K) Staining of electroporated retinal sections with the anti-Brn3a antibody at P8 when RGCs have reached their final location at the retinal surface. Note that the majority of GFP expressing cells located at the RGC layer co-localize with Brn3a (yellow cells), indicating that they are RGCs. Scale bar: 100 μm. High-magnification of GFP-expressing RGCs double-labelled with anti-Brn3a at P0 (H) and P8 (L). Scale bar: 25 μm RGC, retinal ganglion cell layer; INL, internal nuclear layer; VZ, ventricular zone.
Figure 4
Figure 4
The timing of electroporation affects the area of gene targeting. (A) In flattened whole mount E16 retinas electroporated with GFP-plasmids at E13, the cells in the central retina are targeted (green cells). In contrast, when electroporation is performed at E14 the location of GFP-expressing cells varies depending on the position of the electrodes. Examples of retinas electroporated at E14 in the medial dorso-nasal region (B) medial ventronasal (C) or the very peripheral ventronasal retina (D). d, dorsal; n, nasal; t, temporal; v, ventral. Scale bar: 500 μm.
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