Gene delivery into mouse retinal ganglion cells by in utero electroporation
© Garcia-Frigola et al; licensee BioMed Central Ltd. 2007
Received: 11 May 2007
Accepted: 17 September 2007
Published: 17 September 2007
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).
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.
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.
Cells in the retina are packaged into highly ordered anatomical layers each with a specialized function. During eye development, the cells in the ventricular zone undergo successive periods of proliferation and differentiation to generate seven types of retinal cells in a precise order (six types of neurons and one glial cell type). Neurogenesis occurs in the mouse retina between E13 and P10, with exit from the cell cycle reaching a maximum at E18-P0 . Postmitotic retinal cells are generated in an highly ordered fashion from the pool of cycling progenitors, with ganglion, amacrine and horizontal cells originating first and rods, bipolar and Müller glia cells differentiating last.
Among the different cell types in the retina, ganglion cells (RGCs) are the only ones whose axons leave the retina, transmitting visual information to the brain. Once RGCs differentiate, their axons exit the eye through the optic disc in the centre of the retina, and they bundle together to form the optic nerve. Mouse RGC axons grow to the ventral diencephalon and while the majority of them cross the midline and form the optic chiasm, a small proportion of them do not. After sorting in the chiasm, the axons extend dorsally through the optic tracts to reach their principal synaptic targets, the superior colliculi (SC) and the lateral geniculate nucleus (LGN). Visual projections to higher brain targets are organized such that two adjacent RGCs in the retina are connected to two adjacent points in the target field. This topographic arrangement allows a continuous image of the visual field to be projected onto the surface of the target structure.
Gene expression profiles in developing retinal tissue during neurogenesis have been defined recently  and together with efforts to identify genes involved in axon guidance, axon targeting or neural connectivity in the retina, these studies have identified a number of candidate genes that may participate in the development of the visual system. However, functional studies must be performed to fully investigate the specific role of each candidate gene. Gene targeting or transgenesis-based strategies are time-consuming, arduous and expensive procedures, and they require the use of specific promoters that are not always available.
In this article we describe a method to ectopically express genes in RGCs during early stages of embryonic development. Using this approach it is possible to visualize the shape and location of RGCs at different stages of retinal differentiation. Moreover, the projection of RGCs expressing ectopic genes can be monitored along the retinofugal pathway, and even at the optic chiasm, LGN and SC.
Gene delivery into the embryonic retina
Previous studies have demonstrated the versatility of electroporation as a method to deliver DNA into the developing telencephalic vesicles [3–6], as well as into certain retinal cell types in newborn mice . However, gene delivery into the embryonic mammalian retina has not yet been achieved. We have adapted a method for in utero electroporation of cortical neurons , to deliver genes into the mouse retina during development, and more specifically into RGCs and amacrine cells.
Pregnant females were anesthetized and attached with adhesive tape to the cover of a 150 mm plastic Petri-dish by the anterior and posterior limbs. This immobilization facilitated their rotation and improved the injection of the DNA into the eyes of the embryos. The abdominal cavity was then opened and the uterine horns were exposed. Using a graduated pulled glass mouth-micropippette and with the aid of by a portable stereoscope, green fluorescent protein (GFP)-expressing plasmids were injected monocularly through the uterus wall into each embryo. We found it more convenient to use a graduated mouth-micropipette than a picospritzer because it facilitated the rotation of the mother when access to the embryo's eye was restricted, making it difficult to inject the DNA. However, a picospritzer could also be used in cases where very precise volumes must be injected. In the plasmids used, GFP was driven by the CAG promoter (chicken β-actin promoter with CMV enhancer), a very strong and ubiquitous promoter that is similar to those described previously [6, 7]. We also used the cytomegalovirus (CMV) promoter but the levels of GFP expression were lower than those achieved with the CAG promoter.
As expected, the number of GFP expressing cells in the retina varied according to the volume of DNA solution injected. Smaller injection volumes covered a smaller area of the retina and thus, the DNA was accessible to fewer cells. To confirm this hypothesis, electroporations were performed in the retina using different concentrations of DNA in the same volume of (0.2 μg/μl, 0.5 μg/μl, 1 μg/μl and 2 μg/μl). At 0.2 μg/μl no GFP expression was detected, while at the remaining concentrations examined no differences were observed in the area expressing GFP (Data not shown).
Time course of expression in electroporated cells
To determine the proportion of GFP positive cells that were in fact RGCs, retinal sections were labelled with the Islet1/2 antibody. Although the relationship between the onset of Islet1/2 expression and axonogenesis is unknown, this transcription factor is a known marker of RGCs during development . Islet1/2 recognized cells in the most internal layer of the retina, which constitutes the final destination of RGCs during development. On E14, 24 hours after electroporation, GFP-expressing cells displayed a complementary pattern to that of the RGCs labelled with Islet1/2, and no double labelled cells were found (Figure 2A,B,C). However, 72 hours after electroporation GFP-expressing cells were located in a layer containing differentiated RGCs and a few double-labelled cells could be detected (Figure 2D,E,F). Despite the lack of Islet1/2 staining at E16 in the electroporated cells, many axons exiting the retina were visualized at this stage (Figure 2B, see also Figure 5A). Hence, at least some of the electroporated cells appeared to be RGCs.
To test whether we were targeting RGCs, we labelled RGCs retrogradely in embryos that were electroporated at E13 by applying axonal tracers to the optic tract contralateral to the injected eye (Figure 2J). In retinal sections from these animals, several GFP positive cells were filled with dextran indicating that they were indeed RGCs (Figure 2K–O). Interestingly, most of the double-labelled cells were not located in the most internal retinal layer, the future RGC layer. These results are in agreement with previous observations in the ferret retina suggesting that axons may be generated before the parental cell bodies have migrated to their final position .
The cells electroporated at E13 form amacrine cells and RGCs
To determine the final identity of electroporated cells in newborn and P8 retinas we examined the expression of the transcription factor Brn3a, a marker of a large population of RGCs that appear late in development (Figure 3E–K). Accordingly, 70% of electroporated cells located in the RGC layer expressed Brn3a in P8 retinas and hence, corresponded to retinal ganglion cells (Figure 3H,L). Staining for calbindin, a marker for horizontal cells, in newborn retinas also revealed that the few GFP expressing cells located in the ventricular zone were horizontal cells (Figure 3A–D). We cannot rule out the possibility that other retinal cell types were transfected in low numbers when we electroporated at E13 but in our experiments, we failed to detect any other retinal cell type.
According to their final location in the INL and given their appearance, we assume that the rest of electroporated cells belonged to the diverse population of amacrine cells. Indeed, electroporated cells in the RGC layer that did not express Brn3a may either correspond to displaced amacrine cells or to a population of RGCs that do not express Brn3a.
In summary, electroporation at E13 mainly transfects retinal ganglion cells and amacrine cells, as well as a few sporadic horizontal cells. These findings are in accordance with the proposed competence model of retinal neurogenesis in which these cell types are generated simultaneously in the developing retina [1, 11].
The timing of electroporation affects the area of gene expression in the retina
The differences observed in the location of the electroporated cells between E13 and E14 probably reflects the increase in the size of the retina. At E13, the retina is sufficiently small that the DNA injected extends over the entire retina. As a consequence, most of the electroporation-competent cells incorporated the plasmid. Nevertheless, at E16 peripheral cells in retinas electroporated at E13 did not express GFP because newly generated cells had been added to the edges of the retina during this period. In contrast, at E14 the retina was larger and the plasmid DNA injected was accessible to a proportionally smaller area of the retina. Thus, the location of electroporated cells at E14 is primarily driven by the position of the electrodes.
Electroporation of RGCs enables the entire retinofugal pathway to be visualized
The retina is an accessible component of the CNS that has a well-defined cytoarchitecture. Accordingly, it is a structure in which neurogenesis, cell fate specification and the signals that are responsible for promoting cell survival versus programmed cell death during development can be studied in detail. Within the retina, RGCs are the only neurons that send visual information to higher brain areas and their axons project to the midbrain targets, the SC and the LGN. Through numerous studies, molecular signals that direct the topographic information for the precise wiring during development have been identified. Indeed, transcription factors, guidance molecules, extracellular matrix proteins, neurotrophic factors, and cell death regulating factors are known to be involved in the formation of a precise retino-thalamic and retino-collicular map.
Many studies to identify factors that control the development of the visual system have been carried out in the chick, zebrafish and Xenopus. These are animal models in which their extra-uterine development makes it easy to manipulate the embryonic retina. These studies have provided fundamental information on how retinal neurogenesis and topographic retinofugal projections form during embryogenesis. In mammals, time-consuming and complicated gene-targeting approaches are required to elucidate the function of specific molecules in retinal development or to confirm the results obtained in non-mammalian species [12–19]. Here, we have adapted a fast and relatively easy technique to transfer genes into the embryonic retina that will greatly facilitate a wide variety of studies on the development of the visual system in mammals.
The vertebrate neural retina is patterned along all its axes, and retinal differentiation initiates in the central retina, expanding to the periphery . The position of each RGC along the anterior-posterior (A-P) and dorsal-ventral (D-V) axes (its positional identity) determines its connectivity and is crucial for the formation of the topographic maps. Our method for in utero retinal electroporation permits the ectopic expression of genes along the central-peripheral, dorso-ventral and anterior-posterior axes of the embryonic retina. Hence, this technique enables the position of the electroporated RGC cell bodies to be precisely matched with the location of their axonal projections in the LGN and SC.
Cell-fate determination and axonal navigation are achieved by the combinatorial activity of multiple transcription factors and axon guidance molecules . The procedure described here may be used to promote the simultaneous expression of several genes in gain-of-function experiments, or to downregulate genes of interest by electroporation of specific iRNA's.
Studies of neurogenesis can also benefit from this in utero electroporation technique. The six types of retinal neurons are generated in a very orderly manner that is conserved across all species. Ganglion, amacrine and horizontal cells differentiate first, from E11 to P0 in the mouse, and bipolar cells, Müller glia and rods are produced last . Electroporation in neonatal mice leads to the expression of ectopic genes mostly in rods and in a small number of bipolar and Müller glia cells . While other authors have reported some electroporation of RGCs in the adult retina [22, 23], we and others have failed to reproduce this result . However, we have accomplished highly efficient gene delivery to RGCs during embryogenesis. One possible explanation for our results is that cycling cells are the more receptive to electroporation during development. During embryogenesis, all progenitor cells in the ventricular zone could be electroporated, while only those cells that exit the cell cycle shortly after electroporation (RGCs and amacrine cells) will take up sufficient plasmid to be visualized. Indeed, in targeted progenitors that continue cycling the plasmid incorporated will be diluted out over the divisions later and the levels of GFP would become too low to be detected. This hypothesis is supported by our results showing that ganglion, amacrine and a few horizontal cells are electroporated at E13, since all these cell types are generated simultaneously during development [1, 11]. Although it has been suggested that a number of cone cells also differentiate during this period, a significant number of transfected cones was not detected in our experiments. One possible explanation for this is that the production of cones takes place in a very restricted time window, just before or after our electroporation experiments were performed. Indeed, although birth dating of most retinal types has been investigated intensely, cone differentiation is poorly understood. It is significant that in a recent study, GFP labeled RGC or horizontal cells were not detected when the retina of newborn mice was electroporated, but they did detect GFP labeled cone cells (Matsuda and Cepko, 2004). Thus, perhaps the time at which cones are born does not adhere strictly to that described in much earlier studies .
The fact that Islet1/2 positive cells do not express GFP 24 hours post-electroporation once again suggests that post-mitotic cells are not targeted. In addition, this theory is also consistent with studies demonstrating the electroporation of RGCs in P1 ferrets , particularly because in this organism the peak of RGC generation occurs between P3 and P6 .
We have described here a technique for efficient gene delivery into RGCs by in utero electroporation. Using this approach, cells at different stages of retinal differentiation, as well as the entire retinofugal pathway, can be visualized by GFP fluorescence. The use of this technique will benefit research into different aspects of nervous system development including neurogenesis, axon guidance, patterning of axon projections, synaptogenesis, refinement of connections and visual processing.
Animals and DNA constructs
All the animals used in this study were pigmented C57BL6/J pregnant females, since pigmented eyes are easier targets for DNA injection through the uterus wall. Although CD1 albino embryos were also successfully electroporated, the use of pigmented mice increased the efficiency of the protocol. Animals were treated in accordance with Spanish and European Union guidelines for the Care and Use of Laboratory Animals.
A plasmid containing the GFP coding sequence under the control of the CAG promoter (pCAG-GFP) was used in all the electroporation experiments performed here, similar to that previously described [6, 7]. Plasmid DNA was purified using a conventional midiprep kit (Qiagen, Valencia CA) and resuspended in TE. DNA was generally injected in a 1 μg/μl solution.
In utero electroporation
Timed-pregnant C57BL6/J females were anesthetized by intraperitoneal delivery of sodium pentobarbital (0.625 mg per 10 g body weight) and Rytodrine (0.1 ml of a 14 mg/ml solution). The abdomen was opened and the uterine horns exposed. The DNA solution (0.2 μg/μl, 0.5 μg/μl, 1 μg/μl or 2 μg/μl + 0.03% fast green in PBS) was injected into one eye of each embryo using a graduated pulled-glass micropipette. The head of each embryo was placed between tweezer-type electrodes (CUY650-P5 Nepa GENE, Chiba, Japan) and five square electric pulses (38 V, 50 ms) were passed at 1 s intervals using an electroporator (CUY21E, Nepa GENE). The wall and skin of the abdominal cavity were sutured and closed, and the embryos were allowed to develop normally.
Immunostaining of retinal sections and visualization of the retinofugal pathway
Electroporated embryos or pups were perfused and fixed in 4% paraformaldehyde (PFA) for 1–3 days. The heads were cryopreserved in 30% sucrose in PBS and then embedded in Cryoblock (Labonord). Cryosections (30 μm) were obtained and non-specific binding was blocked in 5% Normal goat serum (NGS) in PBS + 0.2% Tween-20 for 1 h at room temperature. The sections were incubated overnight with the anti-Islet1/2 antibody (diluted 1:10000, a generous gift of Dr. Tom Jessell and Susan Morton, Columbia University), the anti-Calbindin antibody (1:2000, Swant), or anti Brn3a (1:200, Chemicon) diluted in 1% NGS-PBS-0.2% Tween-20 at 4°C. A Cy3-conjugated rat anti-guinea pig (Jackson Immunoresearch), Alexa-Fluor-546-goat anti-rabbit (Molecular Probes) and Alexa-Fluor 546-rabbit anti-mouse were used as secondary antibodies, respectively.
To visualize retinal projections at different levels of the retinofugal pathway, the brains were fixed in 4% PFA, embedded in 3% agarose and sectioned in a vibratome (100 μm). The hypothalamus, LGN and SC areas were photographed by conventional microscopy.
Quantification of P8 retinas
Retinal sections from mice were analyzed (n = 2) and a total number of 398 cells were included in the study. Each given percentage was calculated as the average percentage ± SEM
Dextran retrograde labelling of RGC axons
Embryos were electroporated with CAG-GFP at E13, sacrificed at E17, and the embryo's heads were dissected out, the palate removed and the optic chiasm exposed. Dextran tetramethyl rhodamine (MW 6000, Molecular probes) was applied to the contralateral optic tract for retrograde transport to the retina. Heads were maintained in carbonated ACSF buffer for 4 hours at RT, in cold ACSF overnight and fixed in 4% paraformaldehyde the next morning. Retinal sections (100 μm) were obtained using a vibratome
We are grateful to Angel Barco, Carol Mason and Tim Petros for their critically reading of the manuscript. We thank Robert Hindges for technical advice at the beginning of this work, and Tom Jessell and Susan Morton for the Islet 1/2 antibody. This work was supported by grants to E. H. from Human Frontiers Science Program (CDA-0023) and from the Spanish Government (BFU-2004-0058). E.H. is a Ramón y Cajal Investigator from the Consejo Superior de Investigaciones Científicas (CSIC).
- Cepko CL, Austin CP, Yang X, Alexiades M, Ezzeddine D: Cell fate determination in the vertebrate retina. Proc Natl Acad Sci U S A. 1996, 93 (2): 589-595. 10.1073/pnas.93.2.589.PubMed CentralView ArticlePubMedGoogle Scholar
- Blackshaw S, Harpavat S, Trimarchi J, Cai L, Huang H, Kuo WP, Weber G, Lee K, Fraioli RE, Cho SH, Yung R, Asch E, Ohno-Machado L, Wong WH, Cepko CL: Genomic analysis of mouse retinal development. PLoS Biol. 2004, 2 (9): E247-10.1371/journal.pbio.0020247.PubMed CentralView ArticlePubMedGoogle Scholar
- Bai J, Ramos RL, Ackman JB, Thomas AM, Lee RV, LoTurco JJ: RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci. 2003, 6 (12): 1277-1283. 10.1038/nn1153.View ArticlePubMedGoogle Scholar
- Inoue T, Krumlauf R: An impulse to the brain--using in vivo electroporation. Nat Neurosci. 2001, 4 Suppl: 1156-1158. 10.1038/nn1101-1156.View ArticlePubMedGoogle Scholar
- Tabata H, Nakajima K: Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neurosci. 2001, 103 (4): 865-872. 10.1016/S0306-4522(01)00016-1.View ArticleGoogle Scholar
- Borrell V, Yoshimura Y, Callaway EM: Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J Neurosci Methods. 2005, 143 (2): 151-158. 10.1016/j.jneumeth.2004.09.027.View ArticlePubMedGoogle Scholar
- Matsuda T, Cepko CL: Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A. 2004, 101 (1): 16-22. 10.1073/pnas.2235688100.PubMed CentralView ArticlePubMedGoogle Scholar
- Saito T, Nakatsuji N: Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol. 2001, 240 (1): 237-246. 10.1006/dbio.2001.0439.View ArticlePubMedGoogle Scholar
- Brown A, Yates PA, Burrola P, Ortuno D, Vaidya A, Jessell TM, Pfaff SL, O'Leary DD, Lemke G: Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell. 2000, 102 (1): 77-88. 10.1016/S0092-8674(00)00012-X.View ArticlePubMedGoogle Scholar
- Johnson JK, Casagrande VA: Prenatal development of axon outgrowth and connectivity in the ferret visual system. Vis Neurosci. 1993, 10 (1): 117-130.View ArticlePubMedGoogle Scholar
- Livesey FJ, Cepko CL: Vertebrate neural cell-fate determination: lessons from the retina. Nat Rev Neurosci. 2001, 2 (2): 109-118. 10.1038/35053522.View ArticlePubMedGoogle Scholar
- Herrera E, Brown LY, Aruga J, Rachel RA, Dolen G, Mikoshiba K, Brown S, Mason CA: Zic2 patterns binocular vision by specifying the uncrossed retinal projection. Cell. 2003, 114 (5): 545-557. 10.1016/S0092-8674(03)00684-6.View ArticlePubMedGoogle Scholar
- Herrera E, Marcus R, Li S, Williams SE, Erskine L, Lai E, Mason C: Foxd1 is required for proper formation of the optic chiasm. Development. 2004, 131 (22): 5727-5739. 10.1242/dev.01431.View ArticlePubMedGoogle Scholar
- Pak W, Hindges R, Lim YS, Pfaff SL, O'Leary D D: Magnitude of Binocular Vision Controlled by Islet-2 Repression of a Genetic Program that Specifies Laterality of Retinal Axon Pathfinding. Cell. 2004, 119 (4): 567-578. 10.1016/j.cell.2004.10.026.View ArticlePubMedGoogle Scholar
- Erkman L, Yates PA, McLaughlin T, McEvilly RJ, Whisenhunt T, O'Connell SM, Krones AI, Kirby MA, Rapaport DH, Bermingham JR, O'Leary DD, Rosenfeld MG: A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system. Neuron. 2000, 28 (3): 779-792. 10.1016/S0896-6273(00)00153-7.View ArticlePubMedGoogle Scholar
- Williams SE, Mann F, Erskine L, Sakurai T, Wei S, Rossi DJ, Gale NW, Holt CE, Mason CA, Henkemeyer M: Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic chiasm. Neuron. 2003, 39 (6): 919-935. 10.1016/j.neuron.2003.08.017.View ArticlePubMedGoogle Scholar
- Pratt T: The winged helix transcription factor Foxg1 facilitates retinal ganglion cell axon crossing of the ventral midline in the mouse. Development. 2004, 131: 3773-3784. 10.1242/dev.01246.View ArticlePubMedGoogle Scholar
- Huberman AD, Dehay C, Berland M, Chalupa LM, Kennedy H: Early and rapid targeting of eye-specific axonal projections to the dorsal lateral geniculate nucleus in the fetal macaque. J Neurosci. 2005, 25 (16): 4014-4023. 10.1523/JNEUROSCI.4292-04.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Pfeiffenberger C, Cutforth T, Woods G, Yamada J, Renteria RC, Copenhagen DR, Flanagan JG, Feldheim DA: Ephrin-As and neural activity are required for eye-specific patterning during retinogeniculate mapping. Nat Neurosci. 2005, 8 (8): 1022-1027. 10.1038/nn1508.PubMed CentralView ArticlePubMedGoogle Scholar
- Holt CE, Harris WA: Position, guidance, and mapping in the developing visual system. J Neurobiol. 1993, 24 (10): 1400-1422. 10.1002/neu.480241011.View ArticlePubMedGoogle Scholar
- Jessell TM: Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet. 2000, 1 (1): 20-29. 10.1038/35049541.View ArticlePubMedGoogle Scholar
- Dezawa M, Takano M, Negishi H, Mo X, Oshitari T, Sawada H: Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach. Micron. 2002, 33 (1): 1-6. 10.1016/S0968-4328(01)00002-6.View ArticlePubMedGoogle Scholar
- Kachi S, Oshima Y, Esumi N, Kachi M, Rogers B, Zack DJ, Campochiaro PA: Nonviral ocular gene transfer. Gene Ther. 2005, 12 (10): 843-851. 10.1038/sj.gt.3302475.View ArticlePubMedGoogle Scholar
- Carter-Dawson LD, LaVail MM: Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine. J Comp Neurol. 1979, 188 (2): 263-272. 10.1002/cne.901880205.View ArticlePubMedGoogle Scholar
- Huberman AD, Murray KD, Warland DK, Feldheim DA, Chapman B: Ephrin-As mediate targeting of eye-specific projections to the lateral geniculate nucleus. Nat Neurosci. 2005, 8 (8): 1013-1021. 10.1038/nn1505.PubMed CentralView ArticlePubMedGoogle Scholar
- Henderson Z: Development of ganglion cell topography in ferret retina. J Neurosci. 1988, 8 (4): 1994-1205.Google Scholar
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