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Retinal ganglion cell differentiation in cultured mouse retinal explants
Methods 28 (2002) 448–456 www.elsevier.com/locate/ymeth
Retinal ganglion cell differentiation in cultured mouse retinal explants Steven W. Wang,a Xiuqian Mu,a William J. Bowers,b and William H. Kleina,* a
Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA b Center for Aging and Developmental Biology and Department of Neurology, University of Rochester, Rochester, NY 14642, USA Accepted 4 September 2002
Abstract The availability of genetically engineered mice harboring specific mutations in genes affecting one or more retinal cell types affords new opportunities for investigating the genetic regulatory mechanisms of vertebrate retina formation. When identifying critical regulatory genes involved in retina development it is often advantageous to complement in vivo analysis with in vitro characterization. In particular, by combining classical techniques of retinal explant culturing with gene transfer procedures relying on herpes simple virus (HSV) amplicon vectors, gain-of-function analysis with genes of interest can be performed quickly and efficiently. Here, details are provided for isolating and culturing explants containing retinal progenitor cells and for infecting the explants with HSV expression vectors that perturb or rescue retinal ganglion cells, the first cell type to differentiate in the retina. In addition, the availability of sensitive techniques to monitor gene expression, including detection of reporter gene expression using antibodies and detection of endogenous marker gene expression using quantitative RT-PCR, provides an effective means for comparing wild-type and mutant retinas from genetically engineered mice. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Genetically engineered mice; Mouse retinal explants; Herpes simplex virus expression vectors; Retinal ganglion cell gene expression
1. Introduction Because of its unique position and highly organized structure, the vertebrate retina serves as an important model for investigating fundamental questions with regard to genetic regulatory mechanisms in central nervous system (CNS) development [1]. The recent availability of numerous genetically engineered mice harboring targeted mutations affecting retinal development has made the mouse an attractive organism for the study of retinal gene expression and function. Production of different cell types in the mouse retina occurs in two major waves [1–4]. The first wave peaks at E14.5, giving rise to early forming cell types, including retinal ganglion cells (RGCs), cone photoreceptors, amacrine cells, and horizontal cells. The second wave peaks at P4 giving rise to late-forming cell types, including rod photoreceptors, bipolar cells, and M€ uller glial cells (Fig. 1). The differentiation of individual retinal cell types can *
Corresponding author. Fax: 1-713-790-0329. E-mail address: [email protected] (W.H. Klein).
be monitored over developmental time in wild-type and mutant retinas using a variety of techniques and marker genes. One particularly useful approach is that of culturing retinal tissue isolated from early embryonic stages where the neural retina largely consists of undifferentiated neural epithelial progenitor cells. When cultured under the appropriate conditions, the explanted tissue recapitulates retinal development and retinal cell type differentiation with high fidelity. This is the case for normal retinal development and for abnormal development caused by targeted mutations in critical retinal regulatory genes. Retinal explants can be readily infected with herpes simplex virus (HSV) amplicon vectors to determine the effects of overexpression or misexpression of genes of interest. Our laboratory has been particularly interested in the transcription factors that are critical for RGC commitment and differentiation. Using mutant retinas that lacked either the POU domain transcription factor Brn3b (Pou4f2) or the proneural bHLH transcription factor Math5, we have been able to directly observe the effects of loss-of-function and gain-of-function of these factors
1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 2 6 4 - 5
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Fig. 1. Two waves of retinal cell type differentiation. The curves depict the relative number of retinal progenitors that exit the cell cycle and commit to a specific fate over time. RGCs appear at E11 and peak at E13.5 as do amacrine cells, horizontal cells, and cone photoreceptor cells. The other cell types, bipolar cells, M€ uller glia, and rod photoreceptor cells, appear later as indicated. Modified from [2,42].
during RGC differentiation [5–9]. Initiation of RGC formation starts at E11 in the mouse when expression of Math5 is first detectable in a field of progenitor cells within the central retina [10]. Targeted deletion of Math5 (and mutation of its ortholog lakritz in zebrafish) results in a loss of greater than 90% of RGCs, indicating that
Math5 is an essential transcription factor for RGC formation [7,10,11]. Both positive and negative regulatory mechanisms act upstream of Math5 to regulate its expression in progenitor cells. Thus, Pax6 is essential for RGC commitment and Math5 expression [3] while the Notch signaling pathway inhibits Math5 expression
Fig. 2. Genes regulating RGC differentiation. Math5 is essential for establishing the competence of progenitor cells to advance to a RGC fate and for suppressing amacrine cell fate. Other transcription factors may be required to specify RGCs. The Brn3 factors are necessary for the terminal differentiation of RGCs, particularly axon outgrowth. Some of the functions of the Brn3 factors may rely on the activity of other transcription factors, possibly Olf-1, Olf-2, and Irx6 [14].
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through repression by the bHLH factor Hes1 [12]. Hes6 may antagonize Hes1 repression of Math5, suggesting a complex genetic regulatory network [13]. Math5 expression is necessary but not sufficient for the expression of Brn3b, the earliest known marker of RGC differentiation. The lack of Brn3b expression in Math5-deficient retinas is due to the inability of retinal progenitor cells to commit to a RGC fate [7,10]. It appears that only a subset of Math5-positive retinal progenitor cells becomes specified to a RGC fate but the mechanisms regulating the events between Math5 expression and Brn3b activation are unknown (Fig. 2). Brn3b is required for normal terminal RGC differentiation because RGCs lacking Brn3b have defects in axonal outgrowth and pathfinding and most Brn3bdeficient RGCs eventually undergo apoptosis [5,6,14]. GAP-43, a protein known to be associated with axonal outgrowth, is severely downregulated in Brn3b-deficient retinas [8]. Brn3b is closely related to two other POU domain factors that are expressed in differentiating RGCs, Brn3a (Pou4f1), and Brn3c (Pou4f3). In the retina, Brn3c partially compensates for the loss of Brn3b, and Brn3b compensates for the loss of Brn3c, suggesting that these factors have both distinct and overlapping functions in RGC differentiation [9]. The role of Brn3a in the retina remains to be elucidated. In summary, Math5 and Brn3 factors are positioned at central nodes in the genetic hierarchy leading to the differentiation of RGCs. Here, we described the available mouse lines harboring mutant Math5, Brn3b, and Brn3c alleles and detail the methods used to analyze RGC differentiation in vitro using retinal explants isolated from wild-type and mutant mouse embryos.
2. Mice harboring Math5, Brn3a, Brn3b, and Brn3c mutant alleles Table 1 lists the mouse lines currently available in our laboratory that harbor mutant alleles of Math5 and Brn3 factors. Particularly useful are alleles containing
reporter gene knock-ins. To trace the soma of retinal progenitors or differentiating RGCs, we have generated lacZ and green fluorescent protein (GFP) knock-ins. To follow the fate of axonal outgrowth, we have made use of the human placental alkaline phosphatase (AP) gene, which encodes a plasma membrane-associated protein. We have also created a Brn3b–GAP-43 knock-in allele, wherein the Brn3b gene is replaced with a GAP-43–RFP fusion gene to determine whether GAP-43 can rescue Brn3bÕs function in axonal outgrowth. All mouse lines listed in Table 1 are available on request.
3. Procedures for preparing and culturing retinal explants It is important to consider the stage in retinal development to be used for explant culturing. Retinal development is governed by both extrinsic and intrinsic factors and the competency of cells within the retinal explant will change as development proceeds. Thus, the choice of when to isolate the retinal tissue depends on the particular question being addressed. That is, if retinas are isolated at late stages in development, the early differentiation wave will be largely absent. Haverkamp and Wassle [15] provide information on useful retinal cell-specific antibodies. Selection of the culture substrate is also a major consideration. Plastic substrates usually provide good adhesion for coating materials like poly(D -lysine) but plastics are often inadequate for many light microscopy methods. We culture retinal tissue directly on coated plastic petri dishes when the analysis is to be performed by phase-contrast microscopy. If DIC, epifluorescent, or confocal microscopy are involved, a specific glass substrate is used. The type and treatment of coverglass is described in Section 3.1. When culturing retinal explants exogenous serum is usually avoided. Section 3.2 describes two different combinations of serum-free culturing media. Procedures for retinal tissue dissection are detailed in Section 3.3.
Table 1 Genetically engineered mice for investigating RGC differentiation Targeted gene
Reporter
RGC expression
Non-RGC expression
RGC phenotype
Reference
Brn3a (Pou4f1) Brn3b (Pou4f2)
None lacZ, AP, GFP, GAP-43–RFP
All projection neurons All projection neurons
AP
Wild type 70% RGC loss; abnormal axonal growth and pathfinding Wild type
[40] [5,6,14]
Brn3c (Pou4f3)
E13.5 to adult Starts at E12.5, peaks at E15.5, reduced at E18.5 Starts at E14.5
Math5
lacZ, GFP
90% RGC loss
[7,10]
Brn3b:Brn3c
GFP, AP:AP
More severe than Brn3b
[9]
Starts at E11, peaks at E13.5, reduced at E17.5 Same as Brn3b and Brn3c
Inner ear and other projection neurons Subregions of the developing brain All projection neurons
[41]
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3.1. Substrates Most experiments involving cultured retinal explants require relatively sophisticated microscopy. Light penetration and diffraction are therefore critical practical issues. Although poly(D -lysine)-coated thin-bottom plastic petri dishes are commercially available, it is generally more desirable to grow the retinal tissue on glass for better image quality. The type of glass, the cleaning method, and the preparation of coatings are all important for the proper attachment, survival, and maturation of the neurons. Desag coverslips manufactured by Erie Scientific (Portsmouth, NH) are especially suitable for retinal explants and other neuronal cell cultures. Cleaning and etching procedures used in our laboratory are modified specifically for retinal explants from the protocol of Goslin et al. [16] and detailed as follows: (1) Place coverslips (22 22 mm, No. 1 or No. 1.5, depending on the preference for objectives) in ceramic racks (Thomas Scientific; 8542-E40) and rinse in water (2 20 min). (2) Incubate the coverslips in concentrated nitric acid for 24–72 h at room temperature. (This step removes the debris and etches the surface of the coverslips.) (3) Rinse 4 1 h in Milli-Q or comparable-grade water. Tap off excess water. (4) Place racks in beakers, cover with foil, and autoclave with dry cycle for 30 min and drying time of 10 min. (5) Move the beakers to a 37 °C incubator and dry overnight. Do not bake the coverslips; overbaking will prevent the coating from adhering to the glass. (6) In a laminar-flow hood, collect the acid-treated sterile coverslips into petri dishes and seal with Parafilm. The treated coverslips can be stored indefinitely at room temperature. To directly observe the behavior of RGC axons, molecules that provide for axon attraction and adhesion are required on the substrate. Metri-gel provides an excellent growth surface for RGC axons, but it is expensive and difficult to prepare. We routinely use natural mouse laminin isolated from the Engelbreth– Holm–Swarm sarcoma (Invitrogen Life Tech.; 23017015). Laminin is thought to be the basal membrane ligand for the a6 b1 integrin receptor at the RGC axon membrane. Results with laminin have been satisfactory and consistent. The preparation of coating materials and the procedures for coating are detailed as follows: (1) Prepare the laminin coating solution (this and the following steps are performed in a laminar-flow hood): Poly(D -lysine) (BD Biosciences; 35-4210), 100 lg/ml in water; laminin (Invitrogen Life Tech.; 23017-015), 50 lg/ ml. The mixed solution is filtered and 12-ml aliquots are stored at )70 °C. (2) Thaw the laminin–poly(D -lysine) solution at 4 °C on the night prior to use.
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(3) Fill each well of a six-well culture dish (Falcon; 35-3046) with 3 ml autoclaved water. (4) Place one treated coverslip onto the water surface of each well and equilibrate for 3 min. (5) Push the coverslips into the water with a pair of sterile forceps. To release trapped air bubbles underneath the coverslip, lightly tap each coverslip against the bottom with the forceps. (6) Aspirate the water from each well. (7) Add 1 ml of laminin–poly(D -lysine) solution to each well and gently swirl. (8) Wrap the six-well dish with aluminum foil and shake gently on an Orbitron rotator at 4 °C overnight. Be sure that the entire area of the coverslip is immersed in the laminin–poly(D -lysine) solution. (9) The wrapped culture dish can be stored for up to 1 week. Alternatively, the laminin–poly(D -lysine) solution can be recycled for an additional two coatings. (10) To prepare for retinal tissue culture, aspirate the laminin–poly(D -lysine) solution and wash each well 3 with 3 ml sterile deionized water. (11) Add 4 ml of culture medium (see below) to each well containing a laminin–poly(D -lysine)-coated coverslip. The dishes are ready for use. 3.2. Culture medium Culture medium containing serum may greatly interfere with normal differentiation. To objectively investigate the differentiation of retinal cell types including RGCs, a serum-free medium is essential. Two different formulations, DulbeccoÕs modified EagleÕs medium (DMEM; Invitrogen; 21068028) plus N-2 (Invitrogen; 17502048) and Neurobasal plus B-27, are regularly used in our laboratory. DMEM plus N-2 is widely used for neuronal cell culture, particularly short-term culturing. The preparation is as follows: (1) Distribute each N-2 5-ml supplement into 10 equal aliquots. (2) Into each aliquot, add 0.5 ml of penicillin/streptomycin (Invitrogen; 15140122). (3) Into each aliquot, add 0.2 ml of L -glutamine (Cellgro; 25-005-CI) and store the aliquots at )20 °C. (4) Thaw the 1.2-ml aliquots prior to use and transfer the solution to a 50-ml Falcon tube containing 48.8 ml DMEM. (5) In addition to DMEM, the final culture medium contains the following ingredients: bovine insulin (10 lg/ ml), human holotransferrin (200 lg/ml), progesterone (0.12 lg/ml), selenite (0.1 lg/ml), penicillin (100 lg/ml), streptomycin (100 lg/ml), and L -glutamine (120 lg/ml). In DMEM plus N-2 medium, RGC axons begin to degenerate 7 days in culture. The loss of RGC axons may be caused by the high osmolarity and lack of essential growth factors in the medium [17]. Neurobasal plus B-27 is occasionally used in our laboratory when
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prolonged culturing is required. It was originally developed for prolonged low-density cultures of hippocampal neurons and contains 20 balanced components including vitamins E and A, glutathione, pyruvate, catalase, superoxide dismutase, and linolenic acid [18]. The supplement was later modified for RGC regeneration by the addition of several neural growth factors required for the long-term survival of mature RGCs (B. Barres, personal communication). The procedure for preparation of Neurobasal plus N-27 culturing medium is as follows: (1) B-27 supplement (Invitrogen-Gibco; 17504-044) is distributed into 10 equal aliquots of 0.5 ml. (2) Add 0.25 ml of N-2 supplement (Invitrogen; 17502048) to each B-27 aliquot. (3) Add 0.5 ml of penicillin/streptomycin (Invitrogen; 15140122) to each aliquot. (4) Add 0.2 ml of L -glutamine (Cellgro; 25-005-CI) to each aliquot and store the aliquots at )20 °C.
(5) Thaw the 1.45-ml B-27 aliquot at room temperature prior to use and transfer the solution to a 50-ml Falcon tube containing 48 ml of Neurobasal media. (6) Add BDNF (Peprotech; 450-02) to a final concentration of 50 ng/ml. (7) Add CNTF (Peprotech; 450-13) to a final concentration of 10 ng/ml. (8) Add forskolin (Sigma; F-6886) dissolved in dimethyl sulfoxide as a 1000 stock to a final concentration of 50 lM. (9) Use 4 ml of medium in every six-well dish. Renew 50% of the culture medium every 2 days. 3.3. Retina dissection In the mouse, mass production of RGCs begins at E13.5 when RGCs express Brn3b and emit axons [6]. To best study axon outgrowth in newly formed RGCs, retinal dissection should be between E13 and E13.5.
Fig. 3. Dissection of E13.5 retina. (A) An isolated eye is placed in 1 PBS and the tissues surrounding the retina are removed by fine-tip forceps. (B) The remaining structure contains the lens (L) and neural retina. The retinal fissure (arrow) on the ventral side is easily identified at this stage. (C) After removal of the lens with forceps, the retinal tissue is bisected along the fissure and then along the presumptive dorsal–ventral midline using the edge of a steel needle. (D) The bisected retinal tissues are transferred to a culture dish piece by piece using a P200 pipette. The retinal quadrants are placed according to their original positions at specific angles for subsequent recognition (d–t, dorsal–temporal; d–n, dorsal–nasal; v–t, ventral– temporal; v–n, ventral–nasal).
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(The date of plug detection is defined as E0.5.) Embryos are collected from pregnant females following isofluorane anesthesia under IACUC guidelines. Embryos are maintained in 1 phosphate-buffered saline (PBS) (pH 7.4) on ice. One eye of each embryo is isolated and transferred with a pair of tweezers to a fresh dish of icecold PBS. Tissues, including the pigmented epithelial layer surrounding the retina, are removed followed by removal of the lens and vitreous structures under a dissecting microscope. The fissure located on the ventral side of the retina is clearly visible at this stage and can therefore be used as a positional marker. The retina can then be dissected into different regions using a steel needle (Fig. 3). Pieces of the dissected retinal tissue are transferred to a culture dish using a P200 pipette. To ensure RGC axon growth, the retinal tissues are positioned vitreous side down and a slight pressure is applied against the substrate with a steel needle. Dishes containing retinal tissue can then be incubated at 37 °C with 5% CO2 and 99% humidity. The first emitted RGC axons can be observed after 12 h in culture. The retinal explants can either be directly observed under a microscope or be prepared for in situ antibody staining or RNA extraction for RT-PCR. We have found that antibody staining for reporter gene expression (lacZ and AP) rather than substrate staining makes multicolor analysis easier. In our hands, two lacZ antibodies from Cortex Biochem (CR001RP2 and CR001M) work consistently well in either mouse retinal explants or cryo-sectioned retinal tissues. Several antibodies for AP have given good results (Accurate (MEDCLA638), Cortex Biochem (CR2135SP), Chemicon (CBL207)). Although GFP is visible under the fluorescence microscope, low levels of expression or loss of signal during the fixation process often occurs in mouse tissues. GFP antibodies overcome these problems. Chemicon produces a chicken antibody (AB16901) that works well for our preparations.
4. Analysis of RNA expression in the developing retina We have developed sensitive and efficient PCR-based methods for RNA detection in the developing retina in vivo and in vitro. Below, we discuss our procedures for RNA isolation (Section 4.1) and quantitative RT-PCR (Section 4.2). 4.1. Isolation of total RNA from retinal tissue Obtaining high-quality RNA is essential for the analysis of gene expression using RT-PCR methodologies. Although there are several methods (e.g., [19]) and commercial kits available for total RNA isolation from tissues, the most widely used is the Trizol method (Invitrogen). The following is the protocol that we have
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used successfully to isolate high-quality total RNA from mouse retinas: (1) Aliquot 1-ml of Trizol into clean 1.5-ml Eppendorf tubes and place on ice. (2) Dissection of embryonic retina is performed as described in Section 3.3. (3) Vortex vigorously to dissolve the retinal tissue. (4) Add 200 ll of chloroform and mix well by vortexing. (5) Centrifuge at 13,000 rpm for 5 min. Transfer the top aqueous phase to a fresh tube. It is essential not to disturb the interface. (6) Add an equal volume of 2-propanol and mix well. (7) Centrifuge at 13,000 rpm for 10 min. Pellets should be observable. The RNA can be stored as a pellet at )80 °C for prolonged periods. (8) Wash with 70% ethanol. Discard the residual ethanol and air dry for 2 min. Be careful not to overdry because the RNA will be difficult to dissolve. (9) Dissolve the RNA in diethyl pyrocarbonate (DEPC)-treated water. (10) Determine the quantity and quality of the isolated total RNA by UV absorption and electrophoreses of 3–5 lg on a formaldehyde denaturing agarose gel. Typically, 5–10 lg of RNA can be isolated from one liter of E14.5 embryos using the above procedure. Highquality RNA should have an OD260=280 ratio of 1.7–2.0 and display sharp 28S and 18S bands on the agarose gel with a 28S/18S ratio of 2. When the amount of total RNA is limited, the quantity can be examined by a Bioanalyzer (Agilent). 4.2. Quantitative RT-PCR For quantifying mRNA levels, a combination of reverse transcription and PCR is the method of choice. This technique is especially useful for investigating gene expression during retina development. In addition, a high-throughput analysis can be established using a 96or 384-well format, which is suitable for confirming differential expression results from microarray screening. During the early cycles, the PCR product is generally too low to be detected. After a determined number of cycles, the reaction reaches a plateau and further amplification ceases. Thus, the relative input amount of the amplified sequence can be determined accurately only during the middle cycles where amplification is exponential. When performing quantitative PCR, it is important to determine the optimal PCR cycles empirically. The advent of real-time PCR has greatly facilitated the quantitative PCR process because the accumulation of the PCR product is monitored and recorded in real time, making it unnecessary to optimize the PCR cycles. Because of the extremely sensitive nature of PCR, caution should be taken in performing quantitative
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RT-PCR and interpreting the results. Several aspects requiring special attention are listed below: (1) Primer design: primers should be approximately 20 bp with G + C content of 50%. Each primer pair should have a similar Tm value. (2) DNA contamination: RNA isolated by the method described in Section 4.1 is generally free of DNA. Nevertheless, for each new batch of RNA, a reverse transcription negative control should be included. If DNA contamination is an issue, the RNA should be treated with RNase-free DNase I. Effects of DNA contamination can also be minimized by using primers spanning introns. (3) Master mixture: Make a master mixture whenever possible to minimize pipetting errors. (4) Control genes: Include genes whose expression patterns are unaltered by the experimental perturbation. (5) Replication of experiments: Repeat the quantitative RT-PCR with different batches of RNA to minimize systematic errors. The following protocol analyzes the expression of the GAP-43 gene in E14.5 wild-type and Brn3b-deficient retinas to illustrate the method [8]. Brn3b and actin genes serve as controls. Primers: GAP-43 (forward), 50 -GTGCTGCTAAAG CTACT-30 ; GAP-43 (reverse), 50 -CTTCAGAGTGGA GCTGAGAA-30 ; Brn3b (forward), 50 -CAACGGCTCC GGCATGTGC-30 ; Brn3b (reverse), 50 -CTCTTGCTCT GGGCCTCG-30 ; b-actin (forward), 50 -TCTGGAAGC CTACTTCGCCA-30 ; b-actin (reverse), CCGGTTCAC AATCTCTCTGA-30 . First-strand synthesis by reverse transcription: 4 lg total RNA (either wild-type or Brn3b-null RNA); 2 ll oligo(dT); DEPC-treated water to 10 ll. Mix well at 65 °C for 10 min. Add 4 ll 5 RT buffer (250 mM Tris– HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2 ); 2 ll 0.1 M dithiothreitol; 2 ll dNTP (10 mM); 2 ll Superscript II (Invitrogen) to a total volume of 20 ll. Incubate at 42 °C for 2 h. Inactivate the reverse transcriptase by heating at 95 °C for 10 min.
a final concentration of 20 pmol/ll for each primer. Add 2 ll of each primer pair to either a wild-type or a Brn3bnull cDNA-containing tube. Mix well and start the PCR as follows: 95 °C, 15 min to activate the Taq DNA polymerase, followed by the desired cycles of 94 °C, 30 s; 50 °C, 30 s; 72 °C, 40 s. For GAP-43, 25 cycles are used; for Brn3b and b-actin, 20 cycles are used. The reaction is terminated by an extra 5 min of extension and stored at 4 °C. Run 10 ll of each reaction on a 2% agarose gel containing ethidium bromide and visualize the PCR products on a UV light box. It is preferable to run the samples for each gene in adjacent lanes. 4.4. Real-time PCR Real-time PCR requires a specialized instrument available from several manufacturers (Light Cycler System from Roche; Smart Cycler System from Cepheid; iCycler from Bio-Rad; Mx4000 System from Stratagene; Prism7700 from ABI). Setting up the RTPCR is essentially the same as described above, except that for the particular real-time PCR instrument, specific enzymes and probes are required. In real-time PCR, a fluorescent dye (syber green) or specific fluorescent probes (e.g., Taqman probe, Hybridization probe, Molecular beacon) are included in the PCR [20]. Although more expensive, sequence-specific probes add specificity to the assay and enable multiplexing analysis. Even though these probes follow somewhat different principles, they all enable real-time monitoring and recording of the accumulation of the PCR products during the entire PCR process. Expression levels can be calculated by analyzing the shape of the accumulation curve or by determining when the signal raises above a designated threshold value. This eliminates the cumbersome steps of determining the optimal cycles for each gene. Real-time PCR also makes it possible to perform high-throughput gene expression analysis. This is especially useful for confirming data from microarray analysis.
4.3. Quantitative PCR The amount of cDNA to be used in the PCR depends on the expression level of the gene of interest. For most genes, cDNA equivalent to 20 ng of total RNA is sufficient in a 25-ll PCR. Dilute the RT reaction accordingly. HotStart Taq DNA polymerase (Qiagen) is desirable because this procedure eliminates nonspecific amplification. Set up the following 4 master mixture for both wildtype and Brn3b-null cDNA: 20 ll 5 PCR buffer; 8 ll dNTP (2.5 mM each); 4 ll diluted wild-type or Brn3bnull cDNA; 2 ll HotStart Taq; 62 ll water. Aliquot 24 ll of each mixture into three 0.2-ml PCR tubes. Prepare a mix of the forward and reverse primers for each gene to
5. Introduction of ectoptic genes into retinal explants by HSV infection Introducing genes of interest into retinal tissue affords a powerful way to analyze genetic regulatory mechanisms involved in retinal development. We have made extensive use of the HSV amplicon developed by Federoff and colleagues [21] as an effective means of gene transfer into retinal explants. The virion packaging does not require a helper virus so toxicity to the infected cells is minimal [21]. High titers of packaged virus correlate well with high transfection rates into the cultured retinal explant. Below, we describe the HSV vector, production
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Fig. 4. HSV amplicon for gene transfer into retinal explants. (A) The HSV amplicon vector contains a cytomegalovirus (CMV) promoter driving a GFP reporter gene, an ampicillin resistance gene for amplification in Escherichia coli, and HSV-1 origin of replication and packaging sequence. The gene of interest is place under the control of the HSV IE 4/5 promoter. (B) GFP expression demonstrating a high infection efficiency in the E13.5 retinal tissue. An estimated 80% of the cells in the retinal explant are expressing GFP 1 h after viral amplicon incubation.
of replication-defective HSV, and its application to investigations of RGC differentiation. HSV-1 is a naturally neurotropic virus capable of establishing latent infection within neurons, but it also possesses the ability to infect a wide range of cellular targets. The cellular receptors responsible for virion docking and uptake have been cloned, including the herpes virus entry mediators A (HveA) and C (HveC) [22–26]. Not surprisingly, these receptors (or appropriate homologues) are expressed on a variety of cell types. The HSV life cycle involves long periods of latency, and to that end, the virus has evolved a number of elaborate and highly efficient mechanisms to avoid detection and elimination by immune cells [27]. These properties have led to the development of two forms of HSV-1 -based delivery vectors capable of in vivo and in vitro gene transfer to the nervous system recombinant and amplicon vectors [28–31]. The following discourse will detail the attributes of the amplicon platform. The HSV-1 amplicon is a specifically designed eukaryotic expression plasmid containing two noncoding HSV-derived elements: the HSV origin of DNA replication (OriS) and the cleavage/packaging sequence (Fig. 4). This highly versatile plasmid can easily be manipulated to contain desired cis elements and genes of very large sizes (theoretical limit is 150 kb minus the parental amplicon unit) [32–36]. Common to all amplicon vectors, heterogeneous transcription units are cloned into the modified plasmid using conventional molecular cloning techniques and the resultant construct is pack-
aged into enveloped viral particles for subsequent transduction of cells or tissues. Amplicon plasmids are dependent on helper virus function to provide the replication machinery and structural proteins necessary for packaging amplicon vector DNA into viral particles. A replication-defective virus that lacks an essential viral regulatory gene conventionally provides helper packaging function. The final product of helper virus-based packaging contains a mixture of varying proportion of helper and amplicon virions. These stocks are plagued by the inherent cytotoxicity that is derived from the contaminating helper virus. Recently, helper virus-free amplicon packaging methods were developed by providing a packaging-deficient helper virus genome via a set of five overlapping cosmids or by using a bacterial artificial chromosome (HSV-BAC) that encodes the entire HSV genome minus its cognate cleavage/packaging signals [21,37]. Helper virus-free methods of packaging have shown great promise in producing amplicon stocks. The absence of viral genes, especially in virions propagated using helper virus-free methods, results in significantly reduced cytotoxicity and immunogenicity profiles. For example, use of the BAC-based packaging strategy requires the cotransfection of eukaryotic cells that are receptive to HSV propagation (i.e., BHK, Vero) with the HSV-BAC, amplicon DNA, and any accessory HSV genes that enhance amplicon titers [21]. Crude vector lysates are then purified by a series of ultracentrifugation steps and titered by expression or transductionbased methodologies [38]. The titers obtained from
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helper virus-free amplicon packaging range from 107 to 108 transducing particles/ml. To infect the retina explant, the dissected tissues are incubated in 25 ll of virus supernatant plus 100 ll of culture medium at 37 °C for 1 h with gentile agitation before they are plated onto the laminin-coated substrate. The infection rate often reaches 80% of the cells in the retinal tissue as determined by GFP expression (Fig. 4).
6. Conclusion Retinal culturing techniques have been widely used for investigating CNS neuronal differentiation since the late 1950s [39]. The generation of genetically engineered mice with specific retinal defects affords the opportunity to greatly extend what has already been done with cultured retinal explants. The fact that retinal explants recapitulate retinal development in vivo to a large degree makes this technique valuable for characterizing retinal phenotypes associated with particular genetically engineered mice. Moreover, the ability to easily transfer exogenous genes into retinal explants using HSV amplicons and other viral-based vectors and the ability to monitor gene expression patterns by extremely sensitive real-time PCR methods enhance the value of this in vitro analysis.
Acknowledgments We thank Ping Pan and Hongxia Sun for technical assistance. The work was supported by the Robert A. Welch Foundation and by grants from the National Eye Institute (EY11930 and EY13523). References [1] C.L. Cepko, Proc. Natl. Acad. Sci. USA 93 (1996) 589–595. [2] R.W. Young, Anat. Rec. 212 (1985) 199–205. [3] T. Marquardt, R. Ashery-Padan, N. Andrejewski, R. Scardigli, F. Guillemot, P. Gruss, Cell 105 (2001) 43–55. [4] C.L. Cepko, Curr. Opin. Neurobiol. 9 (1999) 37–46. [5] L. Gan, S.W. Wang, Z. Huang, W.H. Klein, Dev. Biol. 210 (1999) 469–480. [6] S.W. Wang, L. Gan, S.E. Martin, W.H. Klein, Mol. Cell. Neurosci. 16 (2000) 141–156. [7] S.W. Wang, B.S. Kim, K. Ding, H. Wang, D. Sun, R.L. Johnson, W.H. Klein, L. Gan, Genes Dev. 25 (2001) 24–29. [8] X. Mu, S. Zhao, R. Pershad, T.F. Hsieh, A. Scarpa, S.W. Wang, R.A. White, P.D. Beremund, T.L. Thomas, L. Gan, W.H. Klein, Nucleic Acids Res. 29 (2001) 4983–4993. [9] S.W. Wang, X. Mu, W.J. Bowers, D.S. Kim, D.J. Plas, M.C. Crair, L. Gan, H.J. Federoff, W.H. Klein, Development 129 (2002) 467–477. [10] N.L. Brown, S. Patel, J. Brzezinski, T. Glaser, Development 128 (2001) 2497–2508.
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Retinal ganglion cell differentiation in cultured mouse retinal explants Steven W. Wang,a Xiuqian Mu,a William J. Bowers,b and William H. Kleina,* a
Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA b Center for Aging and Developmental Biology and Department of Neurology, University of Rochester, Rochester, NY 14642, USA Accepted 4 September 2002
Abstract The availability of genetically engineered mice harboring specific mutations in genes affecting one or more retinal cell types affords new opportunities for investigating the genetic regulatory mechanisms of vertebrate retina formation. When identifying critical regulatory genes involved in retina development it is often advantageous to complement in vivo analysis with in vitro characterization. In particular, by combining classical techniques of retinal explant culturing with gene transfer procedures relying on herpes simple virus (HSV) amplicon vectors, gain-of-function analysis with genes of interest can be performed quickly and efficiently. Here, details are provided for isolating and culturing explants containing retinal progenitor cells and for infecting the explants with HSV expression vectors that perturb or rescue retinal ganglion cells, the first cell type to differentiate in the retina. In addition, the availability of sensitive techniques to monitor gene expression, including detection of reporter gene expression using antibodies and detection of endogenous marker gene expression using quantitative RT-PCR, provides an effective means for comparing wild-type and mutant retinas from genetically engineered mice. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Genetically engineered mice; Mouse retinal explants; Herpes simplex virus expression vectors; Retinal ganglion cell gene expression
1. Introduction Because of its unique position and highly organized structure, the vertebrate retina serves as an important model for investigating fundamental questions with regard to genetic regulatory mechanisms in central nervous system (CNS) development [1]. The recent availability of numerous genetically engineered mice harboring targeted mutations affecting retinal development has made the mouse an attractive organism for the study of retinal gene expression and function. Production of different cell types in the mouse retina occurs in two major waves [1–4]. The first wave peaks at E14.5, giving rise to early forming cell types, including retinal ganglion cells (RGCs), cone photoreceptors, amacrine cells, and horizontal cells. The second wave peaks at P4 giving rise to late-forming cell types, including rod photoreceptors, bipolar cells, and M€ uller glial cells (Fig. 1). The differentiation of individual retinal cell types can *
Corresponding author. Fax: 1-713-790-0329. E-mail address: [email protected] (W.H. Klein).
be monitored over developmental time in wild-type and mutant retinas using a variety of techniques and marker genes. One particularly useful approach is that of culturing retinal tissue isolated from early embryonic stages where the neural retina largely consists of undifferentiated neural epithelial progenitor cells. When cultured under the appropriate conditions, the explanted tissue recapitulates retinal development and retinal cell type differentiation with high fidelity. This is the case for normal retinal development and for abnormal development caused by targeted mutations in critical retinal regulatory genes. Retinal explants can be readily infected with herpes simplex virus (HSV) amplicon vectors to determine the effects of overexpression or misexpression of genes of interest. Our laboratory has been particularly interested in the transcription factors that are critical for RGC commitment and differentiation. Using mutant retinas that lacked either the POU domain transcription factor Brn3b (Pou4f2) or the proneural bHLH transcription factor Math5, we have been able to directly observe the effects of loss-of-function and gain-of-function of these factors
1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 2 6 4 - 5
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Fig. 1. Two waves of retinal cell type differentiation. The curves depict the relative number of retinal progenitors that exit the cell cycle and commit to a specific fate over time. RGCs appear at E11 and peak at E13.5 as do amacrine cells, horizontal cells, and cone photoreceptor cells. The other cell types, bipolar cells, M€ uller glia, and rod photoreceptor cells, appear later as indicated. Modified from [2,42].
during RGC differentiation [5–9]. Initiation of RGC formation starts at E11 in the mouse when expression of Math5 is first detectable in a field of progenitor cells within the central retina [10]. Targeted deletion of Math5 (and mutation of its ortholog lakritz in zebrafish) results in a loss of greater than 90% of RGCs, indicating that
Math5 is an essential transcription factor for RGC formation [7,10,11]. Both positive and negative regulatory mechanisms act upstream of Math5 to regulate its expression in progenitor cells. Thus, Pax6 is essential for RGC commitment and Math5 expression [3] while the Notch signaling pathway inhibits Math5 expression
Fig. 2. Genes regulating RGC differentiation. Math5 is essential for establishing the competence of progenitor cells to advance to a RGC fate and for suppressing amacrine cell fate. Other transcription factors may be required to specify RGCs. The Brn3 factors are necessary for the terminal differentiation of RGCs, particularly axon outgrowth. Some of the functions of the Brn3 factors may rely on the activity of other transcription factors, possibly Olf-1, Olf-2, and Irx6 [14].
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through repression by the bHLH factor Hes1 [12]. Hes6 may antagonize Hes1 repression of Math5, suggesting a complex genetic regulatory network [13]. Math5 expression is necessary but not sufficient for the expression of Brn3b, the earliest known marker of RGC differentiation. The lack of Brn3b expression in Math5-deficient retinas is due to the inability of retinal progenitor cells to commit to a RGC fate [7,10]. It appears that only a subset of Math5-positive retinal progenitor cells becomes specified to a RGC fate but the mechanisms regulating the events between Math5 expression and Brn3b activation are unknown (Fig. 2). Brn3b is required for normal terminal RGC differentiation because RGCs lacking Brn3b have defects in axonal outgrowth and pathfinding and most Brn3bdeficient RGCs eventually undergo apoptosis [5,6,14]. GAP-43, a protein known to be associated with axonal outgrowth, is severely downregulated in Brn3b-deficient retinas [8]. Brn3b is closely related to two other POU domain factors that are expressed in differentiating RGCs, Brn3a (Pou4f1), and Brn3c (Pou4f3). In the retina, Brn3c partially compensates for the loss of Brn3b, and Brn3b compensates for the loss of Brn3c, suggesting that these factors have both distinct and overlapping functions in RGC differentiation [9]. The role of Brn3a in the retina remains to be elucidated. In summary, Math5 and Brn3 factors are positioned at central nodes in the genetic hierarchy leading to the differentiation of RGCs. Here, we described the available mouse lines harboring mutant Math5, Brn3b, and Brn3c alleles and detail the methods used to analyze RGC differentiation in vitro using retinal explants isolated from wild-type and mutant mouse embryos.
2. Mice harboring Math5, Brn3a, Brn3b, and Brn3c mutant alleles Table 1 lists the mouse lines currently available in our laboratory that harbor mutant alleles of Math5 and Brn3 factors. Particularly useful are alleles containing
reporter gene knock-ins. To trace the soma of retinal progenitors or differentiating RGCs, we have generated lacZ and green fluorescent protein (GFP) knock-ins. To follow the fate of axonal outgrowth, we have made use of the human placental alkaline phosphatase (AP) gene, which encodes a plasma membrane-associated protein. We have also created a Brn3b–GAP-43 knock-in allele, wherein the Brn3b gene is replaced with a GAP-43–RFP fusion gene to determine whether GAP-43 can rescue Brn3bÕs function in axonal outgrowth. All mouse lines listed in Table 1 are available on request.
3. Procedures for preparing and culturing retinal explants It is important to consider the stage in retinal development to be used for explant culturing. Retinal development is governed by both extrinsic and intrinsic factors and the competency of cells within the retinal explant will change as development proceeds. Thus, the choice of when to isolate the retinal tissue depends on the particular question being addressed. That is, if retinas are isolated at late stages in development, the early differentiation wave will be largely absent. Haverkamp and Wassle [15] provide information on useful retinal cell-specific antibodies. Selection of the culture substrate is also a major consideration. Plastic substrates usually provide good adhesion for coating materials like poly(D -lysine) but plastics are often inadequate for many light microscopy methods. We culture retinal tissue directly on coated plastic petri dishes when the analysis is to be performed by phase-contrast microscopy. If DIC, epifluorescent, or confocal microscopy are involved, a specific glass substrate is used. The type and treatment of coverglass is described in Section 3.1. When culturing retinal explants exogenous serum is usually avoided. Section 3.2 describes two different combinations of serum-free culturing media. Procedures for retinal tissue dissection are detailed in Section 3.3.
Table 1 Genetically engineered mice for investigating RGC differentiation Targeted gene
Reporter
RGC expression
Non-RGC expression
RGC phenotype
Reference
Brn3a (Pou4f1) Brn3b (Pou4f2)
None lacZ, AP, GFP, GAP-43–RFP
All projection neurons All projection neurons
AP
Wild type 70% RGC loss; abnormal axonal growth and pathfinding Wild type
[40] [5,6,14]
Brn3c (Pou4f3)
E13.5 to adult Starts at E12.5, peaks at E15.5, reduced at E18.5 Starts at E14.5
Math5
lacZ, GFP
90% RGC loss
[7,10]
Brn3b:Brn3c
GFP, AP:AP
More severe than Brn3b
[9]
Starts at E11, peaks at E13.5, reduced at E17.5 Same as Brn3b and Brn3c
Inner ear and other projection neurons Subregions of the developing brain All projection neurons
[41]
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3.1. Substrates Most experiments involving cultured retinal explants require relatively sophisticated microscopy. Light penetration and diffraction are therefore critical practical issues. Although poly(D -lysine)-coated thin-bottom plastic petri dishes are commercially available, it is generally more desirable to grow the retinal tissue on glass for better image quality. The type of glass, the cleaning method, and the preparation of coatings are all important for the proper attachment, survival, and maturation of the neurons. Desag coverslips manufactured by Erie Scientific (Portsmouth, NH) are especially suitable for retinal explants and other neuronal cell cultures. Cleaning and etching procedures used in our laboratory are modified specifically for retinal explants from the protocol of Goslin et al. [16] and detailed as follows: (1) Place coverslips (22 22 mm, No. 1 or No. 1.5, depending on the preference for objectives) in ceramic racks (Thomas Scientific; 8542-E40) and rinse in water (2 20 min). (2) Incubate the coverslips in concentrated nitric acid for 24–72 h at room temperature. (This step removes the debris and etches the surface of the coverslips.) (3) Rinse 4 1 h in Milli-Q or comparable-grade water. Tap off excess water. (4) Place racks in beakers, cover with foil, and autoclave with dry cycle for 30 min and drying time of 10 min. (5) Move the beakers to a 37 °C incubator and dry overnight. Do not bake the coverslips; overbaking will prevent the coating from adhering to the glass. (6) In a laminar-flow hood, collect the acid-treated sterile coverslips into petri dishes and seal with Parafilm. The treated coverslips can be stored indefinitely at room temperature. To directly observe the behavior of RGC axons, molecules that provide for axon attraction and adhesion are required on the substrate. Metri-gel provides an excellent growth surface for RGC axons, but it is expensive and difficult to prepare. We routinely use natural mouse laminin isolated from the Engelbreth– Holm–Swarm sarcoma (Invitrogen Life Tech.; 23017015). Laminin is thought to be the basal membrane ligand for the a6 b1 integrin receptor at the RGC axon membrane. Results with laminin have been satisfactory and consistent. The preparation of coating materials and the procedures for coating are detailed as follows: (1) Prepare the laminin coating solution (this and the following steps are performed in a laminar-flow hood): Poly(D -lysine) (BD Biosciences; 35-4210), 100 lg/ml in water; laminin (Invitrogen Life Tech.; 23017-015), 50 lg/ ml. The mixed solution is filtered and 12-ml aliquots are stored at )70 °C. (2) Thaw the laminin–poly(D -lysine) solution at 4 °C on the night prior to use.
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(3) Fill each well of a six-well culture dish (Falcon; 35-3046) with 3 ml autoclaved water. (4) Place one treated coverslip onto the water surface of each well and equilibrate for 3 min. (5) Push the coverslips into the water with a pair of sterile forceps. To release trapped air bubbles underneath the coverslip, lightly tap each coverslip against the bottom with the forceps. (6) Aspirate the water from each well. (7) Add 1 ml of laminin–poly(D -lysine) solution to each well and gently swirl. (8) Wrap the six-well dish with aluminum foil and shake gently on an Orbitron rotator at 4 °C overnight. Be sure that the entire area of the coverslip is immersed in the laminin–poly(D -lysine) solution. (9) The wrapped culture dish can be stored for up to 1 week. Alternatively, the laminin–poly(D -lysine) solution can be recycled for an additional two coatings. (10) To prepare for retinal tissue culture, aspirate the laminin–poly(D -lysine) solution and wash each well 3 with 3 ml sterile deionized water. (11) Add 4 ml of culture medium (see below) to each well containing a laminin–poly(D -lysine)-coated coverslip. The dishes are ready for use. 3.2. Culture medium Culture medium containing serum may greatly interfere with normal differentiation. To objectively investigate the differentiation of retinal cell types including RGCs, a serum-free medium is essential. Two different formulations, DulbeccoÕs modified EagleÕs medium (DMEM; Invitrogen; 21068028) plus N-2 (Invitrogen; 17502048) and Neurobasal plus B-27, are regularly used in our laboratory. DMEM plus N-2 is widely used for neuronal cell culture, particularly short-term culturing. The preparation is as follows: (1) Distribute each N-2 5-ml supplement into 10 equal aliquots. (2) Into each aliquot, add 0.5 ml of penicillin/streptomycin (Invitrogen; 15140122). (3) Into each aliquot, add 0.2 ml of L -glutamine (Cellgro; 25-005-CI) and store the aliquots at )20 °C. (4) Thaw the 1.2-ml aliquots prior to use and transfer the solution to a 50-ml Falcon tube containing 48.8 ml DMEM. (5) In addition to DMEM, the final culture medium contains the following ingredients: bovine insulin (10 lg/ ml), human holotransferrin (200 lg/ml), progesterone (0.12 lg/ml), selenite (0.1 lg/ml), penicillin (100 lg/ml), streptomycin (100 lg/ml), and L -glutamine (120 lg/ml). In DMEM plus N-2 medium, RGC axons begin to degenerate 7 days in culture. The loss of RGC axons may be caused by the high osmolarity and lack of essential growth factors in the medium [17]. Neurobasal plus B-27 is occasionally used in our laboratory when
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prolonged culturing is required. It was originally developed for prolonged low-density cultures of hippocampal neurons and contains 20 balanced components including vitamins E and A, glutathione, pyruvate, catalase, superoxide dismutase, and linolenic acid [18]. The supplement was later modified for RGC regeneration by the addition of several neural growth factors required for the long-term survival of mature RGCs (B. Barres, personal communication). The procedure for preparation of Neurobasal plus N-27 culturing medium is as follows: (1) B-27 supplement (Invitrogen-Gibco; 17504-044) is distributed into 10 equal aliquots of 0.5 ml. (2) Add 0.25 ml of N-2 supplement (Invitrogen; 17502048) to each B-27 aliquot. (3) Add 0.5 ml of penicillin/streptomycin (Invitrogen; 15140122) to each aliquot. (4) Add 0.2 ml of L -glutamine (Cellgro; 25-005-CI) to each aliquot and store the aliquots at )20 °C.
(5) Thaw the 1.45-ml B-27 aliquot at room temperature prior to use and transfer the solution to a 50-ml Falcon tube containing 48 ml of Neurobasal media. (6) Add BDNF (Peprotech; 450-02) to a final concentration of 50 ng/ml. (7) Add CNTF (Peprotech; 450-13) to a final concentration of 10 ng/ml. (8) Add forskolin (Sigma; F-6886) dissolved in dimethyl sulfoxide as a 1000 stock to a final concentration of 50 lM. (9) Use 4 ml of medium in every six-well dish. Renew 50% of the culture medium every 2 days. 3.3. Retina dissection In the mouse, mass production of RGCs begins at E13.5 when RGCs express Brn3b and emit axons [6]. To best study axon outgrowth in newly formed RGCs, retinal dissection should be between E13 and E13.5.
Fig. 3. Dissection of E13.5 retina. (A) An isolated eye is placed in 1 PBS and the tissues surrounding the retina are removed by fine-tip forceps. (B) The remaining structure contains the lens (L) and neural retina. The retinal fissure (arrow) on the ventral side is easily identified at this stage. (C) After removal of the lens with forceps, the retinal tissue is bisected along the fissure and then along the presumptive dorsal–ventral midline using the edge of a steel needle. (D) The bisected retinal tissues are transferred to a culture dish piece by piece using a P200 pipette. The retinal quadrants are placed according to their original positions at specific angles for subsequent recognition (d–t, dorsal–temporal; d–n, dorsal–nasal; v–t, ventral– temporal; v–n, ventral–nasal).
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(The date of plug detection is defined as E0.5.) Embryos are collected from pregnant females following isofluorane anesthesia under IACUC guidelines. Embryos are maintained in 1 phosphate-buffered saline (PBS) (pH 7.4) on ice. One eye of each embryo is isolated and transferred with a pair of tweezers to a fresh dish of icecold PBS. Tissues, including the pigmented epithelial layer surrounding the retina, are removed followed by removal of the lens and vitreous structures under a dissecting microscope. The fissure located on the ventral side of the retina is clearly visible at this stage and can therefore be used as a positional marker. The retina can then be dissected into different regions using a steel needle (Fig. 3). Pieces of the dissected retinal tissue are transferred to a culture dish using a P200 pipette. To ensure RGC axon growth, the retinal tissues are positioned vitreous side down and a slight pressure is applied against the substrate with a steel needle. Dishes containing retinal tissue can then be incubated at 37 °C with 5% CO2 and 99% humidity. The first emitted RGC axons can be observed after 12 h in culture. The retinal explants can either be directly observed under a microscope or be prepared for in situ antibody staining or RNA extraction for RT-PCR. We have found that antibody staining for reporter gene expression (lacZ and AP) rather than substrate staining makes multicolor analysis easier. In our hands, two lacZ antibodies from Cortex Biochem (CR001RP2 and CR001M) work consistently well in either mouse retinal explants or cryo-sectioned retinal tissues. Several antibodies for AP have given good results (Accurate (MEDCLA638), Cortex Biochem (CR2135SP), Chemicon (CBL207)). Although GFP is visible under the fluorescence microscope, low levels of expression or loss of signal during the fixation process often occurs in mouse tissues. GFP antibodies overcome these problems. Chemicon produces a chicken antibody (AB16901) that works well for our preparations.
4. Analysis of RNA expression in the developing retina We have developed sensitive and efficient PCR-based methods for RNA detection in the developing retina in vivo and in vitro. Below, we discuss our procedures for RNA isolation (Section 4.1) and quantitative RT-PCR (Section 4.2). 4.1. Isolation of total RNA from retinal tissue Obtaining high-quality RNA is essential for the analysis of gene expression using RT-PCR methodologies. Although there are several methods (e.g., [19]) and commercial kits available for total RNA isolation from tissues, the most widely used is the Trizol method (Invitrogen). The following is the protocol that we have
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used successfully to isolate high-quality total RNA from mouse retinas: (1) Aliquot 1-ml of Trizol into clean 1.5-ml Eppendorf tubes and place on ice. (2) Dissection of embryonic retina is performed as described in Section 3.3. (3) Vortex vigorously to dissolve the retinal tissue. (4) Add 200 ll of chloroform and mix well by vortexing. (5) Centrifuge at 13,000 rpm for 5 min. Transfer the top aqueous phase to a fresh tube. It is essential not to disturb the interface. (6) Add an equal volume of 2-propanol and mix well. (7) Centrifuge at 13,000 rpm for 10 min. Pellets should be observable. The RNA can be stored as a pellet at )80 °C for prolonged periods. (8) Wash with 70% ethanol. Discard the residual ethanol and air dry for 2 min. Be careful not to overdry because the RNA will be difficult to dissolve. (9) Dissolve the RNA in diethyl pyrocarbonate (DEPC)-treated water. (10) Determine the quantity and quality of the isolated total RNA by UV absorption and electrophoreses of 3–5 lg on a formaldehyde denaturing agarose gel. Typically, 5–10 lg of RNA can be isolated from one liter of E14.5 embryos using the above procedure. Highquality RNA should have an OD260=280 ratio of 1.7–2.0 and display sharp 28S and 18S bands on the agarose gel with a 28S/18S ratio of 2. When the amount of total RNA is limited, the quantity can be examined by a Bioanalyzer (Agilent). 4.2. Quantitative RT-PCR For quantifying mRNA levels, a combination of reverse transcription and PCR is the method of choice. This technique is especially useful for investigating gene expression during retina development. In addition, a high-throughput analysis can be established using a 96or 384-well format, which is suitable for confirming differential expression results from microarray screening. During the early cycles, the PCR product is generally too low to be detected. After a determined number of cycles, the reaction reaches a plateau and further amplification ceases. Thus, the relative input amount of the amplified sequence can be determined accurately only during the middle cycles where amplification is exponential. When performing quantitative PCR, it is important to determine the optimal PCR cycles empirically. The advent of real-time PCR has greatly facilitated the quantitative PCR process because the accumulation of the PCR product is monitored and recorded in real time, making it unnecessary to optimize the PCR cycles. Because of the extremely sensitive nature of PCR, caution should be taken in performing quantitative
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RT-PCR and interpreting the results. Several aspects requiring special attention are listed below: (1) Primer design: primers should be approximately 20 bp with G + C content of 50%. Each primer pair should have a similar Tm value. (2) DNA contamination: RNA isolated by the method described in Section 4.1 is generally free of DNA. Nevertheless, for each new batch of RNA, a reverse transcription negative control should be included. If DNA contamination is an issue, the RNA should be treated with RNase-free DNase I. Effects of DNA contamination can also be minimized by using primers spanning introns. (3) Master mixture: Make a master mixture whenever possible to minimize pipetting errors. (4) Control genes: Include genes whose expression patterns are unaltered by the experimental perturbation. (5) Replication of experiments: Repeat the quantitative RT-PCR with different batches of RNA to minimize systematic errors. The following protocol analyzes the expression of the GAP-43 gene in E14.5 wild-type and Brn3b-deficient retinas to illustrate the method [8]. Brn3b and actin genes serve as controls. Primers: GAP-43 (forward), 50 -GTGCTGCTAAAG CTACT-30 ; GAP-43 (reverse), 50 -CTTCAGAGTGGA GCTGAGAA-30 ; Brn3b (forward), 50 -CAACGGCTCC GGCATGTGC-30 ; Brn3b (reverse), 50 -CTCTTGCTCT GGGCCTCG-30 ; b-actin (forward), 50 -TCTGGAAGC CTACTTCGCCA-30 ; b-actin (reverse), CCGGTTCAC AATCTCTCTGA-30 . First-strand synthesis by reverse transcription: 4 lg total RNA (either wild-type or Brn3b-null RNA); 2 ll oligo(dT); DEPC-treated water to 10 ll. Mix well at 65 °C for 10 min. Add 4 ll 5 RT buffer (250 mM Tris– HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2 ); 2 ll 0.1 M dithiothreitol; 2 ll dNTP (10 mM); 2 ll Superscript II (Invitrogen) to a total volume of 20 ll. Incubate at 42 °C for 2 h. Inactivate the reverse transcriptase by heating at 95 °C for 10 min.
a final concentration of 20 pmol/ll for each primer. Add 2 ll of each primer pair to either a wild-type or a Brn3bnull cDNA-containing tube. Mix well and start the PCR as follows: 95 °C, 15 min to activate the Taq DNA polymerase, followed by the desired cycles of 94 °C, 30 s; 50 °C, 30 s; 72 °C, 40 s. For GAP-43, 25 cycles are used; for Brn3b and b-actin, 20 cycles are used. The reaction is terminated by an extra 5 min of extension and stored at 4 °C. Run 10 ll of each reaction on a 2% agarose gel containing ethidium bromide and visualize the PCR products on a UV light box. It is preferable to run the samples for each gene in adjacent lanes. 4.4. Real-time PCR Real-time PCR requires a specialized instrument available from several manufacturers (Light Cycler System from Roche; Smart Cycler System from Cepheid; iCycler from Bio-Rad; Mx4000 System from Stratagene; Prism7700 from ABI). Setting up the RTPCR is essentially the same as described above, except that for the particular real-time PCR instrument, specific enzymes and probes are required. In real-time PCR, a fluorescent dye (syber green) or specific fluorescent probes (e.g., Taqman probe, Hybridization probe, Molecular beacon) are included in the PCR [20]. Although more expensive, sequence-specific probes add specificity to the assay and enable multiplexing analysis. Even though these probes follow somewhat different principles, they all enable real-time monitoring and recording of the accumulation of the PCR products during the entire PCR process. Expression levels can be calculated by analyzing the shape of the accumulation curve or by determining when the signal raises above a designated threshold value. This eliminates the cumbersome steps of determining the optimal cycles for each gene. Real-time PCR also makes it possible to perform high-throughput gene expression analysis. This is especially useful for confirming data from microarray analysis.
4.3. Quantitative PCR The amount of cDNA to be used in the PCR depends on the expression level of the gene of interest. For most genes, cDNA equivalent to 20 ng of total RNA is sufficient in a 25-ll PCR. Dilute the RT reaction accordingly. HotStart Taq DNA polymerase (Qiagen) is desirable because this procedure eliminates nonspecific amplification. Set up the following 4 master mixture for both wildtype and Brn3b-null cDNA: 20 ll 5 PCR buffer; 8 ll dNTP (2.5 mM each); 4 ll diluted wild-type or Brn3bnull cDNA; 2 ll HotStart Taq; 62 ll water. Aliquot 24 ll of each mixture into three 0.2-ml PCR tubes. Prepare a mix of the forward and reverse primers for each gene to
5. Introduction of ectoptic genes into retinal explants by HSV infection Introducing genes of interest into retinal tissue affords a powerful way to analyze genetic regulatory mechanisms involved in retinal development. We have made extensive use of the HSV amplicon developed by Federoff and colleagues [21] as an effective means of gene transfer into retinal explants. The virion packaging does not require a helper virus so toxicity to the infected cells is minimal [21]. High titers of packaged virus correlate well with high transfection rates into the cultured retinal explant. Below, we describe the HSV vector, production
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Fig. 4. HSV amplicon for gene transfer into retinal explants. (A) The HSV amplicon vector contains a cytomegalovirus (CMV) promoter driving a GFP reporter gene, an ampicillin resistance gene for amplification in Escherichia coli, and HSV-1 origin of replication and packaging sequence. The gene of interest is place under the control of the HSV IE 4/5 promoter. (B) GFP expression demonstrating a high infection efficiency in the E13.5 retinal tissue. An estimated 80% of the cells in the retinal explant are expressing GFP 1 h after viral amplicon incubation.
of replication-defective HSV, and its application to investigations of RGC differentiation. HSV-1 is a naturally neurotropic virus capable of establishing latent infection within neurons, but it also possesses the ability to infect a wide range of cellular targets. The cellular receptors responsible for virion docking and uptake have been cloned, including the herpes virus entry mediators A (HveA) and C (HveC) [22–26]. Not surprisingly, these receptors (or appropriate homologues) are expressed on a variety of cell types. The HSV life cycle involves long periods of latency, and to that end, the virus has evolved a number of elaborate and highly efficient mechanisms to avoid detection and elimination by immune cells [27]. These properties have led to the development of two forms of HSV-1 -based delivery vectors capable of in vivo and in vitro gene transfer to the nervous system recombinant and amplicon vectors [28–31]. The following discourse will detail the attributes of the amplicon platform. The HSV-1 amplicon is a specifically designed eukaryotic expression plasmid containing two noncoding HSV-derived elements: the HSV origin of DNA replication (OriS) and the cleavage/packaging sequence (Fig. 4). This highly versatile plasmid can easily be manipulated to contain desired cis elements and genes of very large sizes (theoretical limit is 150 kb minus the parental amplicon unit) [32–36]. Common to all amplicon vectors, heterogeneous transcription units are cloned into the modified plasmid using conventional molecular cloning techniques and the resultant construct is pack-
aged into enveloped viral particles for subsequent transduction of cells or tissues. Amplicon plasmids are dependent on helper virus function to provide the replication machinery and structural proteins necessary for packaging amplicon vector DNA into viral particles. A replication-defective virus that lacks an essential viral regulatory gene conventionally provides helper packaging function. The final product of helper virus-based packaging contains a mixture of varying proportion of helper and amplicon virions. These stocks are plagued by the inherent cytotoxicity that is derived from the contaminating helper virus. Recently, helper virus-free amplicon packaging methods were developed by providing a packaging-deficient helper virus genome via a set of five overlapping cosmids or by using a bacterial artificial chromosome (HSV-BAC) that encodes the entire HSV genome minus its cognate cleavage/packaging signals [21,37]. Helper virus-free methods of packaging have shown great promise in producing amplicon stocks. The absence of viral genes, especially in virions propagated using helper virus-free methods, results in significantly reduced cytotoxicity and immunogenicity profiles. For example, use of the BAC-based packaging strategy requires the cotransfection of eukaryotic cells that are receptive to HSV propagation (i.e., BHK, Vero) with the HSV-BAC, amplicon DNA, and any accessory HSV genes that enhance amplicon titers [21]. Crude vector lysates are then purified by a series of ultracentrifugation steps and titered by expression or transductionbased methodologies [38]. The titers obtained from
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helper virus-free amplicon packaging range from 107 to 108 transducing particles/ml. To infect the retina explant, the dissected tissues are incubated in 25 ll of virus supernatant plus 100 ll of culture medium at 37 °C for 1 h with gentile agitation before they are plated onto the laminin-coated substrate. The infection rate often reaches 80% of the cells in the retinal tissue as determined by GFP expression (Fig. 4).
6. Conclusion Retinal culturing techniques have been widely used for investigating CNS neuronal differentiation since the late 1950s [39]. The generation of genetically engineered mice with specific retinal defects affords the opportunity to greatly extend what has already been done with cultured retinal explants. The fact that retinal explants recapitulate retinal development in vivo to a large degree makes this technique valuable for characterizing retinal phenotypes associated with particular genetically engineered mice. Moreover, the ability to easily transfer exogenous genes into retinal explants using HSV amplicons and other viral-based vectors and the ability to monitor gene expression patterns by extremely sensitive real-time PCR methods enhance the value of this in vitro analysis.
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