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Retrovirus-mediated gene transfer to retinal explants
Methods 28 (2002) 387–395 www.elsevier.com/locate/ymeth
Retrovirus-mediated gene transfer to retinal explants Jun Hatakeyama and Ryoichiro Kageyama* Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan Accepted 4 September 2002
Abstract Neural retina can be isolated from mouse embryos and maintained in culture for 2–3 weeks. In such retinal explant cultures, precursor cells differentiate into neurons and glial cells and form three cellular layers, mimicking well the normal development. This explant culture system is suitable for genetic manipulation, such as retrovirus-mediated gene transfer. Retroviral vectors can efficiently transfer genes into retinal precursors, and the copy of the viral genome is precisely transmitted to the progeny of infected cells. Thus, this is an excellent method to change stably the phenotypes of dividing cells. It has been shown that retroviruses carrying transcription factor genes efficiently change the fates of infected cells. Bicistronic expression by retroviral vectors is useful to test the effects of various combinations of many transcription factors. With this method, the transcriptional codes for retinal cell type specification are now being elucidated. Thus, retrovirus-mediated gene transfer to the retinal explant culture system offers a powerful and unique tool to analyze the molecular mechanism of neural development. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Basic helix–loop–helix; Bicistronic expression; Green fluorescent protein; Homeodomain; Retinal explant; Retrovirus; Transcriptional code
1. Introduction The retina is an ideal model system to investigate the mechanisms of generation of multiple cell types from common precursors [1,2] because it has a relatively simple structure, mimics normal development in isolated explant cultures, and is therefore easy to analyze [3–7]. In the neural retina, seven types of cells (six types of neurons and one type of glial cells) form three cellular layers: the outer nuclear layer (ONL), which contains rod and cone photoreceptors; the inner nuclear layer (INL), which contains bipolar, horizontal, and amacrine interneurons and M€ uller glial cells; and the ganglion cell layer (GCL), which contains ganglion and displaced amacrine cells. These seven types of cells differentiate from common precursors in an order that is generally conserved among many species: ganglion cells first and bipolar and M€ uller glial cells last. This ordered differentiation is also observed in retinal explant cultures prepared from mouse embryos.
*
Corresponding author. Fax: +81-75-751-4807. E-mail address: [email protected] (R. Kageyama).
It has been shown that retinal cell differentiation is controlled by intrinsic cues, such as transcription factors, and by extrinsic signals, such as neurotrophic factors [1,2]. The function of extrinsic signals can be assessed by direct application to retinal explant cultures, whereas that of intrinsic cues should be tested by introducing the genes into retinal cells. For the latter purpose, transgenic mice are often used. However, the efficiency of misexpression of exogenous genes in transgenic mice is variable, and thus many independent transgenic lines should be examined. One alternative and very successful method is gene introduction by a recombinant retrovirus, an infectious vehicle that efficiently transfers genes. Since retroviruses are infectious only to mitotic cells, neural precursor cells are the major targets in the nervous system. After infection, a single DNA copy (proviral form) of the RNA viral genome is stably integrated into the genome of host cells, and this DNA copy is precisely transmitted to the progeny of infected cells. Thus, the retrovirus is suitable for changing stably the phenotypes of dividing cells. Furthermore, the onset of exogenous gene expression can be easily controlled by changing the time of infection. This feature is particularly good to analyze the
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 5 7 - 8
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roles of transcription factors because they have different functions at different stages of retinal development. This method is also suitable for clonal analysis: identification of cell types and numbers derived from single virus-infected cells. While in utero or ex utero embryos can be used for virus infection study, it is much easier to use retinal explant cultures. Here, we describe the methods of retinal explant cultures and retrovirus-mediated gene transfer and their application to analysis of gene functions in retinal development.
2. Materials and methods 2.1. Retroviral vectors The RNA genome (a plus-strand RNA) of a retrovirus contains the following structures: R, U5, packaging signal (w), gag, pol, env, U3, and R (Fig. 1A) [8]. Among these structures, gag, pol, and env encode capsid protein, reverse transcriptase, and viral coat proteins, respectively. The 50 end of the RNA genome has a cap structure while the 30 end has a polyadenylation site, like cellular mRNAs. After infection, the double-strand
DNA copy (a proviral form) is produced from the RNA genome by reverse transcriptase and, during this process, U3 and U5 sequences are added to the 50 and 30 termini, respectively (Fig. 1A). Thus, the proviral form has two repeats of U3–R–U5 at both ends, which are called long terminal repeats (LTRs) (Fig. 1A). Only one copy of the proviral form is integrated into the host genome. This process requires the breakdown of the nuclear membrane, and therefore the proviral form is integrated into only dividing cells [9]. The integrated proviral DNA is precisely transmitted to the progeny of infected cells. The U3 in the 50 -LTR functions as an enhancer/promoter and directs transcription from the R sequence, while the R in the 30 -LTR directs cleavage and polyadenylation at the 30 -end of the R, thereby yielding a plus-strand RNA. This RNA serves both as the viral genome to be packaged into virions and as the template for translation of viral genes (gag, pol). Within the RNA genome, there are also splicing donor and acceptor sites, which make an alternatively spliced form, the template for translation of env. Retroviral vectors are made from the proviral form. They have 50 - and 30 -LTRs and the packaging signal, which is required for packaging the RNA genome into
Fig. 1. Schematic structures of retroviral and proviral genomes and retroviral vectors. (A) Schematic structures of retroviral and proviral genomes. The retroviral RNA genome contains a cap structure and polyadenylation site at the 50 - and 30 -termini, respectively, as well as gag, pol, and env genes. w is a packaging signal. The proviral DNA contains additional U3 and U5 sequences at the 50 - and 30 -termini, respectively. The U3–R–U5 sequences located at both ends are called long terminal repeats (LTRs). (B) Schematic structures of the retroviral vector pCLIG and its derivatives. This vector allows bicistronic expression from the 50 -LTR promoter so that both cDNAs located upstream and downstream of the IRES can be expressed from a single transcript. In addition, pCLIG contains w and a part of gag, which is required for efficient packaging (wþ ). EGFP, enhanced green fluorescent protein. (Top) pCLIG. (Middle) Both gene X and EGFP are expressed. (Bottom) Gene Y is fused with EGFP. Both gene X and EGFP–gene Y fusion are expressed.
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retroviral particles (Fig. 1B). Because the viral genes (gag, pol, env) are not required in cis, they are deleted and exchanged with exogenous genes. Many retroviral vectors, however, carry a part of gag because the signal required for efficient packaging is extended into the gag region ðwþ ) (Fig. 1B) [10]. In this case, the translation initiation codon of gag is mutated to a stop codon to avoid expression of a truncated gag [11]. When these vectors are introduced into packaging cells, which supply the products of gag, pol, and env, the RNA transcribed from the retroviral vectors is packaged into retroviral particles. However, since these vectors lack viral genes, they are replication incompetent and do not propagate from cell to cell. Many retroviral vectors including pCLIG have been developed from Moloney murine leukemia virus. In pCLIG, the cytomegalovirus (CMV) enhancer is introduced into the U3 region of the 50 -LTR (Fig. 1B), which significantly increases production of the viral RNA in packaging cells [12,13]. This vector exhibits an almost 100-fold increase of the viral titer, compared with others that do not carry the CMV enhancer. Transient transfection of pCLIG into packaging cells is sufficient for a titer of >106 cfu/ml, whereas those lacking the CMV enhancer require establishment of permanent virusproducing lines to obtain similar titers. Thus, pCLIG is advantageous for genes that are toxic to packaging cells. It should be noted that the retrovirus CLIG produced from the pCLIG vector does not carry the CMV enhancer because the U3 region of the 50 -LTR is not included in the viral genome (see Fig. 1A). Another feature of pCLIG is that it allows bicistronic expression from the upstream LTR promoter through the internal ribosome entry site (IRES) (Fig. 1B). Both cDNAs located upstream and downstream of the IRES can be expressed from a single transcript. In addition, this vector carries the enhanced green fluorescent protein (EGFP) gene downstream of the IRES, which is useful to identify the virus-infected cells (Fig. 1B). The exogenous genes can be inserted upstream of the IRES or as a fusion gene with EGFP. In addition to EGFP, human placental alkaline phosphatase and b-galactosidase genes are used as histological markers in other vectors [14,15]. 2.2. Generation of retrovirus To generate infectious virus, retroviral vectors should be introduced into packaging cells, which express gag, pol, and env genes. There are two types of packaging cells that express different env genes, the ecotropic type, which is infectious only to rodent, and the amphotropic type, which is infectious to a wider range of species. For most analysis, we use the ecotropic packaging cells w2mp34 [16] for biological safety. For generating viruses with a titer of 106 cfu/ml, transient transfection of viral
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vectors into packaging cells is enough. For higher titers, establishment of permanent virus-producing lines is necessary. Another point to consider for high virus titers is that the inserted cDNAs should be as short as possible because retroviruses have a size limitation (<8 kb) [17]. In addition, if there are AATAAA or similar sequences in the 30 -noncoding region, they would direct cleavage of RNA and decrease the full-length viral RNA, thereby reducing the virus titer. Thus, preferably only the coding region should be inserted into retroviral vectors. Here is our protocol for generation and concentration of retrovirus. All procedures require special care to prevent biological hazard. 2.2.1. Protocol for generation of retrovirus by transient transfection Step 1. (Day 1) Plate 3 105 packaging cells in 10 ml of 10% fetal bovine serum (FBS)/DulbeccoÕs modified EagleÕs medium (DMEM) in each 100-mm dish. We usually prepare 5–10 dishes. Incubate the cells overnight at 37 °C in 5% CO2 . Step 2. (Day 2) Packaging cells should be within 30– 40% confluency. Prepare the following transfection solution per each dish. Retroviral vector DNA 10 lg Culture medium (such as DMEM) without serum 1 ml LipofectAMINE (Invitrogen) 25 ll (LipofectAMINE plus reagents also work well) Step 3. Mix the solution and keep it at room temperature for 45 min. Step 4. Rinse the cells with serum-free DMEM once. Then, add 4 ml of serum-free DMEM to each dish. Step 5. Add the transfection solution of Step 3 to the cells and incubate for 6 h. Step 6. Discard the medium, and add 5 ml of fresh 10% FBS/DMEM to each dish. Step 7. Incubate the cells for 2–3 days. Step 8. Harvest the supernatant, which contains recombinant retrovirus. Filter the virus solution through a 0.45-lm filter. Step 9. Concentrate the filtered virus solution with Centricon Plus-20 (Amicon, Beverly, MA) at 4 °C. For most analyses, 10-fold concentration is enough. Step 10. Divide the virus solution into small aliquots and store them at )80 °C. To minimize the loss of the virus titer, freeze–thaw should be avoided. Step 11. Check the virus titer as follows. 1. Plate cells (such as NIH 3T3) in six-well plates on the previous day. 2. Next day, remove the medium and add the following. Virus solution + serum-containing culture medium 500 ll 4 mg/ml Polybrene 1 ll 3. Incubate for 1–3 h at 37 °C in 5% CO2 .
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4. Add 1.5 ml of serum-containing medium to dilute Polybrene. 5. Incubate at 37 °C in 5% CO2 for 1–2 days. 6. If the virus carries color-marking genes such as b-galactosidase, stain cells with an appropriate staining procedure. Then, count the stained cells (since pairs of stained cells attached to each other are usually derived from single infected cells, count them as one). In the case of CLIG virus, stain cells by immunochemistry with anti-GFP antibody (see below) since EGFP fluorescence alone is not sufficient for identification of infected cells. Calculate the virus titer: cfu/ml ¼ the number of stained cells/virus volume (ml). 2.3. Retinal explant culture Neural retina can be prepared easily from mice at embryonic day 15.5 (E15.5) or later and be used for explant cultures. However, it is difficult to isolate retina earlier than E15.5. The neural retina isolated from mouse embryos initially consists of two layers, the ganglion cell layer, which contains ganglion cells, and the ventricular zone, which contains retinal precursors. Within 2 weeks, however, almost all cells finish neuronal/glial differentiation and form three layers (Fig. 2I). The explant cultures can be continued for three weeks without any significant cell death. This method is also advantageous to examine retinal development of mice with embryonic lethal mutations. Here is our protocol for retinal explant cultures and retroviral infection. Procedures for retroviral infection require special care to prevent biological hazard. 2.4. Protocol for retinal explant cultures and retroviral infection 2.4.1. Solution: Retinal medium 50% Minimum essential medium (MEM)–Hepes (Invitrogen, San Diego, CA) 25% HanksÕ balanced salt solution (Invitrogen) 25% Heat-inactivated horse serum* 5.75 mg/ml glucose 25 U/ml penicillin 25 lg/ml streptomycin 200 lM L -glutamine *Several lots of horse serum should be tested to obtain the best results. 2.4.2. Procedures Step 1. Take eyeballs and wash them with phosphatebuffered saline (PBS). Keep them in PBS on ice. Step 2. Put 20 ll of PBS in a Millicell-CM chamber (0.4 lm culture plate insert, 30 mm diameter; Millipore, Bedford, MA), and transfer one eyeball into the PBS. Step 3. Under a microscope, tear and peel off the sclera and cornea with fine forceps (Figs. 2A and B), and
take out the neural retina (Fig. 2C), which still has a lens. The pigment epithelium is often removed with sclera. Step 4. Then, remove the lens (Fig. 2D) and lay the neural retina down carefully by making several incisions at the margin in the Millicell-CM chamber (Fig. 2E). The retina should be always submerged in PBS. Note that the ganglion cell layer is up. Step 5. Put 1 ml of retinal medium in each well of a six-well plate, and transfer the Millicell-CM chamber of Step 4 to the six-well plate (Fig. 2F). Repeat Steps 1–5 for more retinal samples. Until all samples are ready, keep the plate on ice. If more plates are prepared, keep the sample-containing plate at 34 °C in 5% CO2 . Finish Steps 1–5 within, at most, 1 h. Otherwise, the retina does not grow well in culture, resulting in thin structures. Now, it is ready for retroviral infection. Step 6. Thaw the retroviral solution on ice. Then, add 1/20 volume of 4 mg/ml Polybrene (the final concentration: 0.2 mg/ml). Step 7. Transfer each Millicell-CM chamber containing the retinal explant to a new six-well plate. Then, add the viral solution onto the retinal explants (Fig. 2G). Keep the samples at 34 °C in 5% CO2 . One hour later, add more virus solution, and keep them at 34 °C in 5% CO2 for additional 2 h. Avoid drying the samples. Step 8. Put 1 ml of retinal medium in each well of a new six-well plate, and transfer each Millicell-CM chamber containing the virus-infected explant to the new six-well plate (Fig. 2H). Step 9. Keep the samples at 34 °C in 5% CO2 . Step 10. A half-day later, change the medium. After this, change medium every other day. This culture can be continued for 2–3 weeks. Check the explants by microscopy when medium is changed. The explants should be of uniform transparency. If the transparency is not even, the lamination of the retina may be destroyed. 2.5. Fixation The infected cells can be analyzed by sections or dissociation of cultured explants [18]. The procedures for fixation are as follows. 2.5.1. Protocol for fixation of sections Step 1. Wash the samples in the Millicell-CM chamber with PBS once, and then fix them with 4% paraformaldehyde for 10 min. Step 2. Wash the samples with PBS twice, and then submerge them in 25% sucrose/PBS for 40 min. Step 3. Cut the chamber membrane around the samples. Embed the samples (together with membrane) in OCT compound. Make frozen sections (16 lm thick) for immunochemistry. The sections can be stored at )80 °C for several months. Before immunochemistry,
J. Hatakeyama, R. Kageyama / Methods 28 (2002) 387–395 Fig. 2. Preparation and retroviral infection of retinal explant cultures. (A) Tear the sclera of an eyeball with fine forceps. The eyeball is placed in PBS in a Millicell-CM chamber. (B) Peel off the sclera and cornea. During this procedure, the pigment epithelium is usually removed with sclera. (C) Take out the neural retina. (D) Remove the lens. (E) Lay down the neural retina carefully. The ganglion cell layer is up. (F) Transfer the chamber to a six-well plate which contains 1 ml of retinal medium in each well. Put the six-well plate on ice while preparing other samples. (G) Transfer the chamber to a new six-well plate which does not contain medium. Add the retroviral solution to the retinal explants. (H) After infection, transfer the chamber to a new six-well plate which contains 1 ml of retinal medium in each well. (I) After culture for 2 weeks, sections are made from explants. The retina consists of three cellular layers. L, lens; NR, neural retina; Sc, sclera.
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the sections should be washed with PBS for 5 min 3 to remove the OCT compound. Now, it is ready for immunochemistry.
with PBS three times. Now, they are ready for immunochemistry. 2.6. Immunochemistry
2.5.2. Protocol for fixation of dissociated cells Step 1. Flush PBS to detach the explants from the membrane. Transfer each explant into a 1.5-ml Eppendorf tube. Step 2. Treat each sample with 300–500 ll of 0.05% trypsin–0.53 mM EDTA at 37 °C for 10 min. Step 3. Add an equal volume of 10% FBS/MEM to inactivate trypsin. Step 4. Centrifuge at 1200 rpm for 5 min and remove the supernatant. Step 5. Add 200 ll of 20 lg=ml DNase I/HanksÕ balanced salt solution to the cell pellet and dissociate the cells with a pipette. Then, add 800 ll of the same solution. Step 6. Plate the dissociated cells on poly-D -lysinecoated eight-well Lab-Tec chamber slides (Nalge Nunc). Cells from one whole retina are usually divided into three or four wells. Step 7. Incubate at 37 °C in 5% CO2 for 3 h. Cells are settled down during this incubation. Step 8. Wash cells with PBS once, and then fix with 4% paraformaldehyde for 10 min. After fixation, wash
Since the EGFP fluorescence alone is not strong enough, immunochemistry with anti-GFP antibody should be performed to identify CLIG virus-infected cells. In addition, specific markers are useful for identification of cell types (Table 1). Step 1. Treat the samples with blocking solution (5% normal goat serum, 0.1% Triton X-100 in PBS) for 1 h. Step 2. Incubate the samples at room temperature overnight in the following solution. PBS 1% normal goat serum 0.1% Triton X-100 The primary antibody (see Table 1 for dilution rates) Step 3. Rinse the samples with PBS. Step 4. Incubate the samples at room temperature for 2 h in the following solution. PBS 1% normal goat serum 0.1% Triton X-100 The secondary antibody (see Table 2 for dilution rates)
Table 1 Primary antibodies Primary antibody
Dilution rate
Identified cell types
Supplier
Anti-GFP Anti-Thy1.2 Anti-p75 Anti-HPC1/syntaxin Anti-choline acetyltransferase Anti-calbindin Anti-neurofilament Anti-protein kinase C Anti-rhodopsin Anti-glutamine synthetase Anti-cyclin D3 Anti-vimentin Anti-nestin Anti-cyclin D1 Anti-Myc
1:500 1:250 1:500 1:200 1:100 1:2500 1:1000 1:100 1:2000 1:500 1:100 1:200 1:500 1:200 1:1000
CLIG virus-infected cell Ganglion cell Ganglion cell Amacrine cell Amacrine cell Amacrine cell, horizontal cell Horizontal cell, ganglion cell Bipolar cell Rod photoreceptor M€ uller glial cell M€ uller glial cell M€ uller glial cell, precursor Precursor Precursor Tag
MBL Pharmingen (San Diego, CA) Promega (Madison, WI) Sigma (St. Louis, MO) Chemicon (Temecula, CA) Chemicon Chemicon Amersham (Arlington Heights, IL) LSL Chemicon Santa Cruz (Santa Cruz, CA) Nichirei (Tokyo, Japan) Pharmingen Calbiochem (La Jolla, CA) MBL
Table 2 Secondary antibodies Secondary antibody
Dilution rate
Supplier
FITC-conjugated goat anti-mouse IgG Cy3-conjugated goat anti-mouse IgG Cy3-conjugated goat anti-rabbit IgG FITC-conjugated goat anti-rabbit IgG Biotinylated anti-mouse IgG Biotinylated anti-rabbit IgG
1:200 1:200 1:200 1:200 1:200 1:200
Vector Laboratories (Burlingame, CA) Amersham (Arlington Heights, IL) Amersham Vector Laboratories Vector Laboratories Vector Laboratories
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Step 40 . Alternatively, incubate the samples at room temperature for 2 h in the following solution. PBS 1% normal goat serum 0.1% Triton X-100 Biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (1:200 diluted) Then, wash the samples with PBS and incubate them at room temperature for 1 h in the following solution. PBS 0.1% Triton X-100 FITC–avidin D or Texas red–avidin D (1:1000 diluted) Step 5. Wash the samples in PBS and mount them in VectaShield (Vector Laboratories, Burlingame, CA). Step 6. Take photographs by microscopy.
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3. Results and discussion Retinal explants were prepared from mouse embryos E17.5 and CLIG retrovirus was applied. After 2 weeks of culture, immunohistochemistry with anti-GFP was performed. Under this condition, CLIG-infected precursor cells differentiate into all retinal cell types (Fig. 3). Rod photoreceptors are most abundant (about 80%), while ganglion cells are very few (less than 1%) (Fig. 3B). Other cell types are within 2–8% each of the total virusinfected cells (Fig. 3B). These ratios reflect normal retinal development during this period, when rod genesis is most frequent [19,20]. Since retinal precursors at E17.5 still have the potential to become all cell types, this explant culture system offers a useful tool to analyze the mechanism of retinal cell type specification.
Fig. 3. Infection of the recombinant retrovirus CLIG into retinal explant cultures. (A) Recombinant retrovirus CLIG was applied to retinal explants, which were prepared from E17.5 mouse embryos. After 2 weeks of culture, sections of explants were subjected to immunohistochemistry with antiGFP antibody. Virus-infected cells were identified in all three layers. (B) Quantification of CLIG virus-infected cells. Rods are most abundant while ganglion cells are very few. (C) Higher magnification of CLIG-infected cells. The cell types can be identified by their morphology, location, and specific markers (see Table 1).
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Retroviral vectors efficiently introduce exogenous genes into retinal precursors and thus are suitable for misexpression study [6]. We have been characterizing roles of transcription factors in retinal development. Particularly, we have been focusing on basic helix–loop– helix (bHLH) and homeodomain factors. Misexpression of neuronal bHLH genes such as Mash1, Math3, and NeuroD promotes neuronal differentiation while inhibiting glial differentiation [7,21–24]. In contrast, the bHLH genes Hes1 and Hes5, which antagonize neuronal bHLH genes, inhibit neuronal differentiation and maintain precursors or promote gliogenesis [6,12,25]. Thus, these bHLH genes play an important role in maintenance of precursors and neuronal/glial fate determination. Although neuronal bHLH genes are expressed not only by rods but also by other cell types, the latter cell types are not induced by such bHLH genes, suggesting that bHLH genes alone are not sufficient for generation of specific neuronal subtypes. For example, bHLH genes Mash1 and Math3 are expressed by bipolar cells and, in mice lacking both Mash1 and Math3, bipolar cells are missing [26]. In these mutant mice, the cells that should normally differentiate into bipolar cells adopt the M€ uller glial cell fate, indicating that there is a fate switch from
neurons to glial cells in the absence of the bHLH genes [26]. However, misexpression of Mash1 or Math3 alone does not induce bipolar cell genesis. It has been shown that homeodomain factors are important for neuronal subtype specification. For example, the homeodomain gene Chx10 is expressed by bipolar cells and, in Chx10mutant mice, bipolar cells are missing [27,28]. Although misexpression of Chx10 alone efficiently makes cells in the INL, many of them are M€ uller glial cells, and there are no mature bipolar cells, indicating that Chx10 alone is not sufficient either to generate bipolar cells [23]. Since neither bHLH nor a homeodomain gene alone is sufficient to generate bipolar neurons, we next tried coexpression of both bHLH and homeodomain genes by a single retroviral vector. For this analysis, bHLH genes (Mash1, Math3) were fused with EGFP of the CLIG vector while Chx10 was placed upstream of the IRES (Fig. 4A). This retrovirus efficiently directs dual expression of bHLH and homeodomain genes: more than 99% of the virus-infected cells coexpress both genes (Figs. 4B–D). Application of this virus efficiently generated mature bipolar cells (protein kinase C ðPKCÞþ ) in the retinal explant cultures (Figs. 4E–G, arrows) [23]. Thus, coexpression of Mash1/Math3 and Chx10 is required
Fig. 4. Retroviruses carrying transcription factor genes promote retinal cell type specification. (A) Schematic structure of the retroviral vector pCLIG-Chx10-Math3. The homeodomain gene Chx10 with a myc tag is inserted upstream of the IRES while the bHLH gene Math3 is inserted as a fusion with EGFP. (B–D) Retinal cells were infected with CLIG–Chx10–Math3 virus and dissociated. More than 99% of the virus-infected cells coexpressed Chx10 (Mycþ ) and Math3 (GFPþ ). (E–G) CLIG–Chx10–Math3 virus was applied to retinal explants prepared from E17.5 mouse embryos and, after 2 weeks of culture, the fates of the virus-infected cells were examined by immunochemistry of sections. Cells infected with CLIG– Chx10–Math3 virus efficiently differentiated into bipolar cells (PKCþ , arrows).
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for bipolar cell fate specification. It is likely that Chx10 specifies the layer (INL) identity while Mash1/Math3 induces the neuronal features within the Chx10-specified layer. Similarly, the bHLH genes Math3 and NeuroD are expressed by amacrine cells and, in mice lacking both Math3 and NeuroD, amacrine cells are missing [7,24]. In these mutant mice, the cells that should normally differentiate into amacrine cells mostly adopt the ganglion cell fate, indicating that there is a fate switch from amacrine to ganglion cells [24]. However, misexpression of Math3 and NeuroD alone does not induce amacrine cell genesis. The homeodomain gene Pax6 is also expressed by amacrine cells but, although Pax6 induces INL-specific cells, it alone cannot induce mature neurons [23,24]. In contrast, combinations of bHLH genes (Math3/ NeuroD) and Pax6 efficiently generate amacrine cells [23,24]. It is likely that Pax6 regulates the layer specificity but cannot generate mature neurons, while Math3/ NeuroD induces the neuronal features within the Pax6specified layer. Thus, combinations of bHLH and homeodomain genes are essential for specification of many neuronal subtypes. As shown above, retrovirus-mediated gene transfer to retinal explants offers a powerful method to examine the transcriptional codes for retinal cell type specification. Particularly, because transcription factors exhibit different activities when expressed either alone or together with others, the feature of bicistronic expression by CLIG vectors is advantageous to analyze the effects of various combinations of many transcription factors. Such transcriptional codes will be useful for regeneration of specific subtypes of neurons [29].
Acknowledgments This work was supported by Special Coordination Funds for Promoting Science and Technology and research grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science.
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Retrovirus-mediated gene transfer to retinal explants Jun Hatakeyama and Ryoichiro Kageyama* Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan Accepted 4 September 2002
Abstract Neural retina can be isolated from mouse embryos and maintained in culture for 2–3 weeks. In such retinal explant cultures, precursor cells differentiate into neurons and glial cells and form three cellular layers, mimicking well the normal development. This explant culture system is suitable for genetic manipulation, such as retrovirus-mediated gene transfer. Retroviral vectors can efficiently transfer genes into retinal precursors, and the copy of the viral genome is precisely transmitted to the progeny of infected cells. Thus, this is an excellent method to change stably the phenotypes of dividing cells. It has been shown that retroviruses carrying transcription factor genes efficiently change the fates of infected cells. Bicistronic expression by retroviral vectors is useful to test the effects of various combinations of many transcription factors. With this method, the transcriptional codes for retinal cell type specification are now being elucidated. Thus, retrovirus-mediated gene transfer to the retinal explant culture system offers a powerful and unique tool to analyze the molecular mechanism of neural development. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Basic helix–loop–helix; Bicistronic expression; Green fluorescent protein; Homeodomain; Retinal explant; Retrovirus; Transcriptional code
1. Introduction The retina is an ideal model system to investigate the mechanisms of generation of multiple cell types from common precursors [1,2] because it has a relatively simple structure, mimics normal development in isolated explant cultures, and is therefore easy to analyze [3–7]. In the neural retina, seven types of cells (six types of neurons and one type of glial cells) form three cellular layers: the outer nuclear layer (ONL), which contains rod and cone photoreceptors; the inner nuclear layer (INL), which contains bipolar, horizontal, and amacrine interneurons and M€ uller glial cells; and the ganglion cell layer (GCL), which contains ganglion and displaced amacrine cells. These seven types of cells differentiate from common precursors in an order that is generally conserved among many species: ganglion cells first and bipolar and M€ uller glial cells last. This ordered differentiation is also observed in retinal explant cultures prepared from mouse embryos.
*
Corresponding author. Fax: +81-75-751-4807. E-mail address: [email protected] (R. Kageyama).
It has been shown that retinal cell differentiation is controlled by intrinsic cues, such as transcription factors, and by extrinsic signals, such as neurotrophic factors [1,2]. The function of extrinsic signals can be assessed by direct application to retinal explant cultures, whereas that of intrinsic cues should be tested by introducing the genes into retinal cells. For the latter purpose, transgenic mice are often used. However, the efficiency of misexpression of exogenous genes in transgenic mice is variable, and thus many independent transgenic lines should be examined. One alternative and very successful method is gene introduction by a recombinant retrovirus, an infectious vehicle that efficiently transfers genes. Since retroviruses are infectious only to mitotic cells, neural precursor cells are the major targets in the nervous system. After infection, a single DNA copy (proviral form) of the RNA viral genome is stably integrated into the genome of host cells, and this DNA copy is precisely transmitted to the progeny of infected cells. Thus, the retrovirus is suitable for changing stably the phenotypes of dividing cells. Furthermore, the onset of exogenous gene expression can be easily controlled by changing the time of infection. This feature is particularly good to analyze the
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 5 7 - 8
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roles of transcription factors because they have different functions at different stages of retinal development. This method is also suitable for clonal analysis: identification of cell types and numbers derived from single virus-infected cells. While in utero or ex utero embryos can be used for virus infection study, it is much easier to use retinal explant cultures. Here, we describe the methods of retinal explant cultures and retrovirus-mediated gene transfer and their application to analysis of gene functions in retinal development.
2. Materials and methods 2.1. Retroviral vectors The RNA genome (a plus-strand RNA) of a retrovirus contains the following structures: R, U5, packaging signal (w), gag, pol, env, U3, and R (Fig. 1A) [8]. Among these structures, gag, pol, and env encode capsid protein, reverse transcriptase, and viral coat proteins, respectively. The 50 end of the RNA genome has a cap structure while the 30 end has a polyadenylation site, like cellular mRNAs. After infection, the double-strand
DNA copy (a proviral form) is produced from the RNA genome by reverse transcriptase and, during this process, U3 and U5 sequences are added to the 50 and 30 termini, respectively (Fig. 1A). Thus, the proviral form has two repeats of U3–R–U5 at both ends, which are called long terminal repeats (LTRs) (Fig. 1A). Only one copy of the proviral form is integrated into the host genome. This process requires the breakdown of the nuclear membrane, and therefore the proviral form is integrated into only dividing cells [9]. The integrated proviral DNA is precisely transmitted to the progeny of infected cells. The U3 in the 50 -LTR functions as an enhancer/promoter and directs transcription from the R sequence, while the R in the 30 -LTR directs cleavage and polyadenylation at the 30 -end of the R, thereby yielding a plus-strand RNA. This RNA serves both as the viral genome to be packaged into virions and as the template for translation of viral genes (gag, pol). Within the RNA genome, there are also splicing donor and acceptor sites, which make an alternatively spliced form, the template for translation of env. Retroviral vectors are made from the proviral form. They have 50 - and 30 -LTRs and the packaging signal, which is required for packaging the RNA genome into
Fig. 1. Schematic structures of retroviral and proviral genomes and retroviral vectors. (A) Schematic structures of retroviral and proviral genomes. The retroviral RNA genome contains a cap structure and polyadenylation site at the 50 - and 30 -termini, respectively, as well as gag, pol, and env genes. w is a packaging signal. The proviral DNA contains additional U3 and U5 sequences at the 50 - and 30 -termini, respectively. The U3–R–U5 sequences located at both ends are called long terminal repeats (LTRs). (B) Schematic structures of the retroviral vector pCLIG and its derivatives. This vector allows bicistronic expression from the 50 -LTR promoter so that both cDNAs located upstream and downstream of the IRES can be expressed from a single transcript. In addition, pCLIG contains w and a part of gag, which is required for efficient packaging (wþ ). EGFP, enhanced green fluorescent protein. (Top) pCLIG. (Middle) Both gene X and EGFP are expressed. (Bottom) Gene Y is fused with EGFP. Both gene X and EGFP–gene Y fusion are expressed.
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retroviral particles (Fig. 1B). Because the viral genes (gag, pol, env) are not required in cis, they are deleted and exchanged with exogenous genes. Many retroviral vectors, however, carry a part of gag because the signal required for efficient packaging is extended into the gag region ðwþ ) (Fig. 1B) [10]. In this case, the translation initiation codon of gag is mutated to a stop codon to avoid expression of a truncated gag [11]. When these vectors are introduced into packaging cells, which supply the products of gag, pol, and env, the RNA transcribed from the retroviral vectors is packaged into retroviral particles. However, since these vectors lack viral genes, they are replication incompetent and do not propagate from cell to cell. Many retroviral vectors including pCLIG have been developed from Moloney murine leukemia virus. In pCLIG, the cytomegalovirus (CMV) enhancer is introduced into the U3 region of the 50 -LTR (Fig. 1B), which significantly increases production of the viral RNA in packaging cells [12,13]. This vector exhibits an almost 100-fold increase of the viral titer, compared with others that do not carry the CMV enhancer. Transient transfection of pCLIG into packaging cells is sufficient for a titer of >106 cfu/ml, whereas those lacking the CMV enhancer require establishment of permanent virusproducing lines to obtain similar titers. Thus, pCLIG is advantageous for genes that are toxic to packaging cells. It should be noted that the retrovirus CLIG produced from the pCLIG vector does not carry the CMV enhancer because the U3 region of the 50 -LTR is not included in the viral genome (see Fig. 1A). Another feature of pCLIG is that it allows bicistronic expression from the upstream LTR promoter through the internal ribosome entry site (IRES) (Fig. 1B). Both cDNAs located upstream and downstream of the IRES can be expressed from a single transcript. In addition, this vector carries the enhanced green fluorescent protein (EGFP) gene downstream of the IRES, which is useful to identify the virus-infected cells (Fig. 1B). The exogenous genes can be inserted upstream of the IRES or as a fusion gene with EGFP. In addition to EGFP, human placental alkaline phosphatase and b-galactosidase genes are used as histological markers in other vectors [14,15]. 2.2. Generation of retrovirus To generate infectious virus, retroviral vectors should be introduced into packaging cells, which express gag, pol, and env genes. There are two types of packaging cells that express different env genes, the ecotropic type, which is infectious only to rodent, and the amphotropic type, which is infectious to a wider range of species. For most analysis, we use the ecotropic packaging cells w2mp34 [16] for biological safety. For generating viruses with a titer of 106 cfu/ml, transient transfection of viral
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vectors into packaging cells is enough. For higher titers, establishment of permanent virus-producing lines is necessary. Another point to consider for high virus titers is that the inserted cDNAs should be as short as possible because retroviruses have a size limitation (<8 kb) [17]. In addition, if there are AATAAA or similar sequences in the 30 -noncoding region, they would direct cleavage of RNA and decrease the full-length viral RNA, thereby reducing the virus titer. Thus, preferably only the coding region should be inserted into retroviral vectors. Here is our protocol for generation and concentration of retrovirus. All procedures require special care to prevent biological hazard. 2.2.1. Protocol for generation of retrovirus by transient transfection Step 1. (Day 1) Plate 3 105 packaging cells in 10 ml of 10% fetal bovine serum (FBS)/DulbeccoÕs modified EagleÕs medium (DMEM) in each 100-mm dish. We usually prepare 5–10 dishes. Incubate the cells overnight at 37 °C in 5% CO2 . Step 2. (Day 2) Packaging cells should be within 30– 40% confluency. Prepare the following transfection solution per each dish. Retroviral vector DNA 10 lg Culture medium (such as DMEM) without serum 1 ml LipofectAMINE (Invitrogen) 25 ll (LipofectAMINE plus reagents also work well) Step 3. Mix the solution and keep it at room temperature for 45 min. Step 4. Rinse the cells with serum-free DMEM once. Then, add 4 ml of serum-free DMEM to each dish. Step 5. Add the transfection solution of Step 3 to the cells and incubate for 6 h. Step 6. Discard the medium, and add 5 ml of fresh 10% FBS/DMEM to each dish. Step 7. Incubate the cells for 2–3 days. Step 8. Harvest the supernatant, which contains recombinant retrovirus. Filter the virus solution through a 0.45-lm filter. Step 9. Concentrate the filtered virus solution with Centricon Plus-20 (Amicon, Beverly, MA) at 4 °C. For most analyses, 10-fold concentration is enough. Step 10. Divide the virus solution into small aliquots and store them at )80 °C. To minimize the loss of the virus titer, freeze–thaw should be avoided. Step 11. Check the virus titer as follows. 1. Plate cells (such as NIH 3T3) in six-well plates on the previous day. 2. Next day, remove the medium and add the following. Virus solution + serum-containing culture medium 500 ll 4 mg/ml Polybrene 1 ll 3. Incubate for 1–3 h at 37 °C in 5% CO2 .
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4. Add 1.5 ml of serum-containing medium to dilute Polybrene. 5. Incubate at 37 °C in 5% CO2 for 1–2 days. 6. If the virus carries color-marking genes such as b-galactosidase, stain cells with an appropriate staining procedure. Then, count the stained cells (since pairs of stained cells attached to each other are usually derived from single infected cells, count them as one). In the case of CLIG virus, stain cells by immunochemistry with anti-GFP antibody (see below) since EGFP fluorescence alone is not sufficient for identification of infected cells. Calculate the virus titer: cfu/ml ¼ the number of stained cells/virus volume (ml). 2.3. Retinal explant culture Neural retina can be prepared easily from mice at embryonic day 15.5 (E15.5) or later and be used for explant cultures. However, it is difficult to isolate retina earlier than E15.5. The neural retina isolated from mouse embryos initially consists of two layers, the ganglion cell layer, which contains ganglion cells, and the ventricular zone, which contains retinal precursors. Within 2 weeks, however, almost all cells finish neuronal/glial differentiation and form three layers (Fig. 2I). The explant cultures can be continued for three weeks without any significant cell death. This method is also advantageous to examine retinal development of mice with embryonic lethal mutations. Here is our protocol for retinal explant cultures and retroviral infection. Procedures for retroviral infection require special care to prevent biological hazard. 2.4. Protocol for retinal explant cultures and retroviral infection 2.4.1. Solution: Retinal medium 50% Minimum essential medium (MEM)–Hepes (Invitrogen, San Diego, CA) 25% HanksÕ balanced salt solution (Invitrogen) 25% Heat-inactivated horse serum* 5.75 mg/ml glucose 25 U/ml penicillin 25 lg/ml streptomycin 200 lM L -glutamine *Several lots of horse serum should be tested to obtain the best results. 2.4.2. Procedures Step 1. Take eyeballs and wash them with phosphatebuffered saline (PBS). Keep them in PBS on ice. Step 2. Put 20 ll of PBS in a Millicell-CM chamber (0.4 lm culture plate insert, 30 mm diameter; Millipore, Bedford, MA), and transfer one eyeball into the PBS. Step 3. Under a microscope, tear and peel off the sclera and cornea with fine forceps (Figs. 2A and B), and
take out the neural retina (Fig. 2C), which still has a lens. The pigment epithelium is often removed with sclera. Step 4. Then, remove the lens (Fig. 2D) and lay the neural retina down carefully by making several incisions at the margin in the Millicell-CM chamber (Fig. 2E). The retina should be always submerged in PBS. Note that the ganglion cell layer is up. Step 5. Put 1 ml of retinal medium in each well of a six-well plate, and transfer the Millicell-CM chamber of Step 4 to the six-well plate (Fig. 2F). Repeat Steps 1–5 for more retinal samples. Until all samples are ready, keep the plate on ice. If more plates are prepared, keep the sample-containing plate at 34 °C in 5% CO2 . Finish Steps 1–5 within, at most, 1 h. Otherwise, the retina does not grow well in culture, resulting in thin structures. Now, it is ready for retroviral infection. Step 6. Thaw the retroviral solution on ice. Then, add 1/20 volume of 4 mg/ml Polybrene (the final concentration: 0.2 mg/ml). Step 7. Transfer each Millicell-CM chamber containing the retinal explant to a new six-well plate. Then, add the viral solution onto the retinal explants (Fig. 2G). Keep the samples at 34 °C in 5% CO2 . One hour later, add more virus solution, and keep them at 34 °C in 5% CO2 for additional 2 h. Avoid drying the samples. Step 8. Put 1 ml of retinal medium in each well of a new six-well plate, and transfer each Millicell-CM chamber containing the virus-infected explant to the new six-well plate (Fig. 2H). Step 9. Keep the samples at 34 °C in 5% CO2 . Step 10. A half-day later, change the medium. After this, change medium every other day. This culture can be continued for 2–3 weeks. Check the explants by microscopy when medium is changed. The explants should be of uniform transparency. If the transparency is not even, the lamination of the retina may be destroyed. 2.5. Fixation The infected cells can be analyzed by sections or dissociation of cultured explants [18]. The procedures for fixation are as follows. 2.5.1. Protocol for fixation of sections Step 1. Wash the samples in the Millicell-CM chamber with PBS once, and then fix them with 4% paraformaldehyde for 10 min. Step 2. Wash the samples with PBS twice, and then submerge them in 25% sucrose/PBS for 40 min. Step 3. Cut the chamber membrane around the samples. Embed the samples (together with membrane) in OCT compound. Make frozen sections (16 lm thick) for immunochemistry. The sections can be stored at )80 °C for several months. Before immunochemistry,
J. Hatakeyama, R. Kageyama / Methods 28 (2002) 387–395 Fig. 2. Preparation and retroviral infection of retinal explant cultures. (A) Tear the sclera of an eyeball with fine forceps. The eyeball is placed in PBS in a Millicell-CM chamber. (B) Peel off the sclera and cornea. During this procedure, the pigment epithelium is usually removed with sclera. (C) Take out the neural retina. (D) Remove the lens. (E) Lay down the neural retina carefully. The ganglion cell layer is up. (F) Transfer the chamber to a six-well plate which contains 1 ml of retinal medium in each well. Put the six-well plate on ice while preparing other samples. (G) Transfer the chamber to a new six-well plate which does not contain medium. Add the retroviral solution to the retinal explants. (H) After infection, transfer the chamber to a new six-well plate which contains 1 ml of retinal medium in each well. (I) After culture for 2 weeks, sections are made from explants. The retina consists of three cellular layers. L, lens; NR, neural retina; Sc, sclera.
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the sections should be washed with PBS for 5 min 3 to remove the OCT compound. Now, it is ready for immunochemistry.
with PBS three times. Now, they are ready for immunochemistry. 2.6. Immunochemistry
2.5.2. Protocol for fixation of dissociated cells Step 1. Flush PBS to detach the explants from the membrane. Transfer each explant into a 1.5-ml Eppendorf tube. Step 2. Treat each sample with 300–500 ll of 0.05% trypsin–0.53 mM EDTA at 37 °C for 10 min. Step 3. Add an equal volume of 10% FBS/MEM to inactivate trypsin. Step 4. Centrifuge at 1200 rpm for 5 min and remove the supernatant. Step 5. Add 200 ll of 20 lg=ml DNase I/HanksÕ balanced salt solution to the cell pellet and dissociate the cells with a pipette. Then, add 800 ll of the same solution. Step 6. Plate the dissociated cells on poly-D -lysinecoated eight-well Lab-Tec chamber slides (Nalge Nunc). Cells from one whole retina are usually divided into three or four wells. Step 7. Incubate at 37 °C in 5% CO2 for 3 h. Cells are settled down during this incubation. Step 8. Wash cells with PBS once, and then fix with 4% paraformaldehyde for 10 min. After fixation, wash
Since the EGFP fluorescence alone is not strong enough, immunochemistry with anti-GFP antibody should be performed to identify CLIG virus-infected cells. In addition, specific markers are useful for identification of cell types (Table 1). Step 1. Treat the samples with blocking solution (5% normal goat serum, 0.1% Triton X-100 in PBS) for 1 h. Step 2. Incubate the samples at room temperature overnight in the following solution. PBS 1% normal goat serum 0.1% Triton X-100 The primary antibody (see Table 1 for dilution rates) Step 3. Rinse the samples with PBS. Step 4. Incubate the samples at room temperature for 2 h in the following solution. PBS 1% normal goat serum 0.1% Triton X-100 The secondary antibody (see Table 2 for dilution rates)
Table 1 Primary antibodies Primary antibody
Dilution rate
Identified cell types
Supplier
Anti-GFP Anti-Thy1.2 Anti-p75 Anti-HPC1/syntaxin Anti-choline acetyltransferase Anti-calbindin Anti-neurofilament Anti-protein kinase C Anti-rhodopsin Anti-glutamine synthetase Anti-cyclin D3 Anti-vimentin Anti-nestin Anti-cyclin D1 Anti-Myc
1:500 1:250 1:500 1:200 1:100 1:2500 1:1000 1:100 1:2000 1:500 1:100 1:200 1:500 1:200 1:1000
CLIG virus-infected cell Ganglion cell Ganglion cell Amacrine cell Amacrine cell Amacrine cell, horizontal cell Horizontal cell, ganglion cell Bipolar cell Rod photoreceptor M€ uller glial cell M€ uller glial cell M€ uller glial cell, precursor Precursor Precursor Tag
MBL Pharmingen (San Diego, CA) Promega (Madison, WI) Sigma (St. Louis, MO) Chemicon (Temecula, CA) Chemicon Chemicon Amersham (Arlington Heights, IL) LSL Chemicon Santa Cruz (Santa Cruz, CA) Nichirei (Tokyo, Japan) Pharmingen Calbiochem (La Jolla, CA) MBL
Table 2 Secondary antibodies Secondary antibody
Dilution rate
Supplier
FITC-conjugated goat anti-mouse IgG Cy3-conjugated goat anti-mouse IgG Cy3-conjugated goat anti-rabbit IgG FITC-conjugated goat anti-rabbit IgG Biotinylated anti-mouse IgG Biotinylated anti-rabbit IgG
1:200 1:200 1:200 1:200 1:200 1:200
Vector Laboratories (Burlingame, CA) Amersham (Arlington Heights, IL) Amersham Vector Laboratories Vector Laboratories Vector Laboratories
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Step 40 . Alternatively, incubate the samples at room temperature for 2 h in the following solution. PBS 1% normal goat serum 0.1% Triton X-100 Biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (1:200 diluted) Then, wash the samples with PBS and incubate them at room temperature for 1 h in the following solution. PBS 0.1% Triton X-100 FITC–avidin D or Texas red–avidin D (1:1000 diluted) Step 5. Wash the samples in PBS and mount them in VectaShield (Vector Laboratories, Burlingame, CA). Step 6. Take photographs by microscopy.
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3. Results and discussion Retinal explants were prepared from mouse embryos E17.5 and CLIG retrovirus was applied. After 2 weeks of culture, immunohistochemistry with anti-GFP was performed. Under this condition, CLIG-infected precursor cells differentiate into all retinal cell types (Fig. 3). Rod photoreceptors are most abundant (about 80%), while ganglion cells are very few (less than 1%) (Fig. 3B). Other cell types are within 2–8% each of the total virusinfected cells (Fig. 3B). These ratios reflect normal retinal development during this period, when rod genesis is most frequent [19,20]. Since retinal precursors at E17.5 still have the potential to become all cell types, this explant culture system offers a useful tool to analyze the mechanism of retinal cell type specification.
Fig. 3. Infection of the recombinant retrovirus CLIG into retinal explant cultures. (A) Recombinant retrovirus CLIG was applied to retinal explants, which were prepared from E17.5 mouse embryos. After 2 weeks of culture, sections of explants were subjected to immunohistochemistry with antiGFP antibody. Virus-infected cells were identified in all three layers. (B) Quantification of CLIG virus-infected cells. Rods are most abundant while ganglion cells are very few. (C) Higher magnification of CLIG-infected cells. The cell types can be identified by their morphology, location, and specific markers (see Table 1).
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Retroviral vectors efficiently introduce exogenous genes into retinal precursors and thus are suitable for misexpression study [6]. We have been characterizing roles of transcription factors in retinal development. Particularly, we have been focusing on basic helix–loop– helix (bHLH) and homeodomain factors. Misexpression of neuronal bHLH genes such as Mash1, Math3, and NeuroD promotes neuronal differentiation while inhibiting glial differentiation [7,21–24]. In contrast, the bHLH genes Hes1 and Hes5, which antagonize neuronal bHLH genes, inhibit neuronal differentiation and maintain precursors or promote gliogenesis [6,12,25]. Thus, these bHLH genes play an important role in maintenance of precursors and neuronal/glial fate determination. Although neuronal bHLH genes are expressed not only by rods but also by other cell types, the latter cell types are not induced by such bHLH genes, suggesting that bHLH genes alone are not sufficient for generation of specific neuronal subtypes. For example, bHLH genes Mash1 and Math3 are expressed by bipolar cells and, in mice lacking both Mash1 and Math3, bipolar cells are missing [26]. In these mutant mice, the cells that should normally differentiate into bipolar cells adopt the M€ uller glial cell fate, indicating that there is a fate switch from
neurons to glial cells in the absence of the bHLH genes [26]. However, misexpression of Mash1 or Math3 alone does not induce bipolar cell genesis. It has been shown that homeodomain factors are important for neuronal subtype specification. For example, the homeodomain gene Chx10 is expressed by bipolar cells and, in Chx10mutant mice, bipolar cells are missing [27,28]. Although misexpression of Chx10 alone efficiently makes cells in the INL, many of them are M€ uller glial cells, and there are no mature bipolar cells, indicating that Chx10 alone is not sufficient either to generate bipolar cells [23]. Since neither bHLH nor a homeodomain gene alone is sufficient to generate bipolar neurons, we next tried coexpression of both bHLH and homeodomain genes by a single retroviral vector. For this analysis, bHLH genes (Mash1, Math3) were fused with EGFP of the CLIG vector while Chx10 was placed upstream of the IRES (Fig. 4A). This retrovirus efficiently directs dual expression of bHLH and homeodomain genes: more than 99% of the virus-infected cells coexpress both genes (Figs. 4B–D). Application of this virus efficiently generated mature bipolar cells (protein kinase C ðPKCÞþ ) in the retinal explant cultures (Figs. 4E–G, arrows) [23]. Thus, coexpression of Mash1/Math3 and Chx10 is required
Fig. 4. Retroviruses carrying transcription factor genes promote retinal cell type specification. (A) Schematic structure of the retroviral vector pCLIG-Chx10-Math3. The homeodomain gene Chx10 with a myc tag is inserted upstream of the IRES while the bHLH gene Math3 is inserted as a fusion with EGFP. (B–D) Retinal cells were infected with CLIG–Chx10–Math3 virus and dissociated. More than 99% of the virus-infected cells coexpressed Chx10 (Mycþ ) and Math3 (GFPþ ). (E–G) CLIG–Chx10–Math3 virus was applied to retinal explants prepared from E17.5 mouse embryos and, after 2 weeks of culture, the fates of the virus-infected cells were examined by immunochemistry of sections. Cells infected with CLIG– Chx10–Math3 virus efficiently differentiated into bipolar cells (PKCþ , arrows).
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for bipolar cell fate specification. It is likely that Chx10 specifies the layer (INL) identity while Mash1/Math3 induces the neuronal features within the Chx10-specified layer. Similarly, the bHLH genes Math3 and NeuroD are expressed by amacrine cells and, in mice lacking both Math3 and NeuroD, amacrine cells are missing [7,24]. In these mutant mice, the cells that should normally differentiate into amacrine cells mostly adopt the ganglion cell fate, indicating that there is a fate switch from amacrine to ganglion cells [24]. However, misexpression of Math3 and NeuroD alone does not induce amacrine cell genesis. The homeodomain gene Pax6 is also expressed by amacrine cells but, although Pax6 induces INL-specific cells, it alone cannot induce mature neurons [23,24]. In contrast, combinations of bHLH genes (Math3/ NeuroD) and Pax6 efficiently generate amacrine cells [23,24]. It is likely that Pax6 regulates the layer specificity but cannot generate mature neurons, while Math3/ NeuroD induces the neuronal features within the Pax6specified layer. Thus, combinations of bHLH and homeodomain genes are essential for specification of many neuronal subtypes. As shown above, retrovirus-mediated gene transfer to retinal explants offers a powerful method to examine the transcriptional codes for retinal cell type specification. Particularly, because transcription factors exhibit different activities when expressed either alone or together with others, the feature of bicistronic expression by CLIG vectors is advantageous to analyze the effects of various combinations of many transcription factors. Such transcriptional codes will be useful for regeneration of specific subtypes of neurons [29].
Acknowledgments This work was supported by Special Coordination Funds for Promoting Science and Technology and research grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science.
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