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[5] Avian neuroretina cells in oncogene studies
[5]
AVIAN NEURORETINA CELLS
[5] A v i a n N e u r o r e t i n a
By
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Cells in Oncogene
PATRICIA CRISANTI, BERNARD PESSAC,
and
Studies
GEORGES CALOTHY
There are two main reasons to use cells from the avian embryonic neuroretina (NR) as an experimental system to study the effects of oncogenes on regulation of cell growth and differentiation. First, NR cultures are exclusively composed of neuroectodermal cells: this tissue is not vascularized and does not contain cells of mesenchymal origin. Second, when these cells are maintained in culture, they rapidly stop dividing and express several markers of neural dfferentiation. The NR is part of the central nervous system and is, therefore, of ectodermal origin. It is derived from the optic cup which is an evagination from the wall of the diencephalon. The optic cup consists of an external layer which gives rise to the pigment epithelium and an internal layer which develops into the NR itself (Fig. 1). 12 A schematic representation of the vertebrate NR is shown in Fig. 2. The adult NR is composed of three types of neurons, photoreceptors, bipolar cells, and ganglion cells, and of two types of interneurons, horizontal and amacrine cells and one type of glial cells (MUller cells). These cells are organized in a layered structure together with two synaptic layers: the outer and the inner plexiform layers (Fig. 2). Two main types of synaptic complexes are present in the NR: the nonribbon (conventional synapses that are found in all neural tissues) and the ribbon synapses that are specific of the NR (Fig. 3). 3 Neuroretina Development and Differentiation The avian NR development proceeds through essentially three phases: proliferation of neuroectodermal precursor ceils, lamination of cell strata, and differentiation of postmitotic cells. 4 During early phases of development, immature cells are present across the entire primitive NR, and mitotic cells are visible in the external part adjacent to the future pigment epithelium. At a precise stage of development, cells become postmitotic and migrate toward the inner face of the NR. They then start to differentiate i R. Adler and D. Farber, Eds. " T h e Retina, Part 1: A Model for Cell Biology Studies." Academic Press, Orlando, FL, 1986. 2 N. J. Berrill and G. Karp, " D e v e l o p m e n t . " McGraw-Hill, New York, 1976. P. Crisanti-Combes, A., Privat, B. Pessac, and G. Calothy, Cell Tissue Rex. 185, 159 (1977). 4 C. J. Barnstable, Mol. Neurobiol. 1, 9 (1987).
METHODS IN ENZYMOLOGY.VOL. 254
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Fie. 2. Synaptic contacts between the main types of retinal cells. The upper layer consists of rods and cones. These sensory cells make synaptic contact with their neighboring cells and with bipolar and horizontal cells. These in turn make synaptic contacts with the inner ganglion cells whose axons constitute the optic nerve (from Berrill and Karfa).
when reaching positions that define their corresponding layers. Thymidine incorporation experiments, at various stages of chicken embryonic development, show that withdrawal from the cell cycle follows a posterior --~ anterior gradient: cells from the posterior pole, close to the optic nerve, are the first to withdraw from the cell cycle and to differentiate. Moreover, all cells located within a given region do not exit the cycle synchronously. Precursors of ganglion cells are the first to stop dividing between ED3 (Day 3 of embryonic development) and EDS, followed by precursors of cones and rods which in contrast will differentiate shortly before hatching. At later stages, most horizontal and amacrine cells become postmitotic. Precursors of bipolar and Mfiller cells are the last to exit from cell cycle. Finally, at ED10, the vast majority of chicken NR cells are postmitotic. Synaptogenesis begins between ED10 and E D l l . Figure 4 describes representative steps of NR histogenesis in chicken embryos between ED6 and 1 day after hatching.
FIG. 3. (A) Typical diads and triads arrangement of synaptic ribbons associated with aligned synaptic vesicles. (B) A large presynaptic with a dense matrix containing asynaptic ribbons with vesicles. Identical synaptic junctions are observed in cultured NR cells.
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Only a few reports have described the kinetics of cell cycle withdrawal in embryonic quail NR (QNR) cells. 5 Because the development of the quail proceeds at a faster rate than that of the chicken, both withdrawal from the cell cycle and lamination take place earlier (17 days vs 20 days in the chicken). Investigation of the kinetics and topography of D N A arrest in the embryonic QNR, by bromodeoxyuridine (BrdU) incorporation in replicating D N A (see methods below), led to the following observations (Fig. 5): At ED7 (Fig. 5A), the lamination process has just begun. Only the ganglion cells are organized in a distinct lamina which is not yet separated from the other cells by the plexiform layer. The remaining NR appears as a homogeneous population of elongated cells in the process of migration. At this stage, no labeled nuclei are observed in ganglion cells whereas a number of fluorescent nuclei are homogenously distributed over a wide zone of inorganized cells. At ED9 (Fig. 5B), cells of the presumptive inner nuclear layer (INL) are still elongated and intermingled. Only the inner plexiform layer (IPL) starts to be individualized. The number of S phases is markedly reduced in comparison to ED7 and ED8. Labeled nuclei are localized in the presumptive INL. The posterior pole appears more differentiated and presents a reduced number of S phases as compared to the anterior pole. At ED10 (Fig. 5C), the lamination process is not achieved but the IPL is visible, while the outer segments of photoreceptors are in formation. At this stage, no labeled nuclei are visible in either pole. Cells could not be labeled with BrdU thereafter.
Dissection NR can be dissected from chicken (CNR) or quail (QNR) embryos as early as days 6-7 of incubation. 3 Heads are first immersed in phosphatebuffered saline (PBS) without Cae+/Mg 2÷ (PBS-) and are parted in two along the median axis with fine forceps. Eyes are dissected out, the lenses are discarded, and the NR is carefully separated from the underlying pigment epithelium under a dissecting microscope. The retinas are spread in PBSand the few adhering pieces of pigment epithelium are cut away. Retinas are pooled, washed twice in PBS-, incubated in PBS- for 10 min at 37 °, and then in a 0.25% (w/v) trypsin solution in PBS- for 20 min. After centrifugation at room temperature, NR are washed three times in Eagle's 5 A. A. Moscona and L. Degenstein, Dev. Neurosci. 4, 211 (1981).
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FIG. 5. Detection of S phases during various stages of development: (A) at ED7, (B) ED9, and (C) ED10. Incorporation of BrdU into replicating D N A was detected by indirect immunofluorescence. See text.
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basal medium (BME), without dissociation, and finally, tissues are dissociated by gentle pipetting in BME supplemented with 5-10% fetal calf serum (FCS) and antibiotics (100/zg/ml kanamycin, 100/zg/ml streptomycin, and 100 units/ml penicillin). The cells are centrifuged at 200 g, resuspensed in culture medium, and counted in a hemocytometer. They appear as single cells with a few tiny clumps. One ED7 NR yields about 5 x 106 cells. They are usually seeded at a density of 3 x 105 cells/cm2 and are incubated at 37 ° in a humidified atmosphere [5% (v/v) CO2 in air]. Medium is renewed every second or third day.
Light and Electron Microscopy Studies of NR Cultures Cultures are observed by light microscopy or processed for electron microscopy as follows: they are fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 hr and postfixed in 1% osmium tetroxide in the same buffer for 1 hr. Fixed cells are stained en bloc with uranyl acetate, dehydrated in ethanol-acetate, and fiat-embedded in Araldite. Ultrathin sections are cut in a plane parallel to the substrate. NR dissected from embryos at the onset of the cultures are processed similarly for electron microscopy. After 24 hr, NR cells from 6 to 7-day-old chicken embryos are composed of clusters of small, round cells (seven to eight times smaller than fibroblasts) that are undifferentiated neuroblasts. Two to 3 days later, these small clusters spread out and start to extend processes. Round cells differentiate from very immature cell types, neuroepithelial like at the time of dissociation, into relatively mature neurons. This maturation process is visualized by the development of perikaryal organelles and transformation of the nucleus from a fusiform notched outline, with numerous chromatine clumps, to a regular, spherical shape with an evenly dispersed chromatin and a prominent nucleolus. In addition, processes grow out of these cells, progress with growth cones on the flattened cells, and form a reticular array between groups of round cells. However, it is difficult, if not impossible, to identify most of these neurons. Photoreceptors cells are identifiable because of the presence of ribbon synapses characteristic of cone receptors in the adult retina. Similarly, ganglion cells could be tentatively identified because of their size, larger than that of any other neuron in culture, and of their general morphology, especially because of the presence of an axon. Areas of neuropiles are distributed in the vicinity of neuronal clusters and, sometimes, in the center of such clusters. They more or less mimic the organization of the retina into cellular and plexiform layers. Synapses are found after 8 days in vitro and are still visible after 15
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and 21 days in culture. They correspond to the different types found in the retina in vivo, although they most often lack the typical organization of postsynaptic elements. A second cell type becomes morphologically distinguishable and is composed of fiat, transparent cells that may represent MOiler cells for two reasons. First, they do not show neuronal characteristics. Second, they display some ultrastructural similarities to MOiler cells of the in vivo retina, namely the presence of large arrays of filaments and microtubules, as well as numerous and extensive attachment plates associated with some of the fibrillar arrays (Fig. 6A). In summary, NR cultures obtained from 7-day-old chicken embryos can be maintained as monolayers and are able to differentiate into neuronal and nonneuronal (glial) cells. This differentiation proceeds to the stage at which morphologically mature synapses are found. It, therefore, appears that synaptogenesis primarily depends on the genetic programs of the cells and that complex interactions, found in tridimensional structures, may not be required for the formation of synapses.
Transdifferentiation From the fourth week onward, new phenotypes appear that might be due to transdifferentiation6 of cells that were initially programmed into the neuronal or glial pathways or otherwise derived from uncommitted stem cells. One of the phenotypes is made of small pigmented cells, whereas the second consists of cells that progressively take on the appearance of lentoid bodies (LB). These structures are formed of densely packed refringent cells and of "bottle cells," both of which appear in culture in lens epithelial cells and follow a differentiation pathway similar to that of the lens (Fig. 6C). The LB are always associated with crystallins identical to those present in lens during development or in cultured lens epithelial cells. Crystallins constitute the bulk of lens proteins and, in birds, fall into three major classes (c~,/3, and 8) that are the products of three multigene families.7,~ c~and 8-crystallins are present on day 28 in cultured QNR and progressively increase until day 42. The amounts of a- and 6-crystallins remain stable thereafter. In QNR cultures passaged once after 10 weeks, the levels of c~and 8-crystallins remain elevated. 9
T. S. Okada, Y. Itoh, K. Watanabe, and G. Eguchi, Dev. Biol. 45, 318 (1975). v D. I. De Pomerai, D. J. Pritchard, and R. M. Clayton, Dev. Biol. 60, 416 (1977). 8 R. M. Clayton, I. Thompson, and D. I. De Pomerai, Nature (London) 282, 628 (1979). 9 L. Simonneau, P. Crisanti, A. M. Lorinet, F. Alliot, Y. Courtois, G. Calothy, and B. Pessac, Mol. CelL Biol. 6, 3704 (1986).
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W i t h d r a w a l from Cell Cycle of C u l t u r e d NR Cultures of CNR or Q N R ceils dissected at E D 6 - E D 7 undergo two or three doublings at the level of cell population under the culture conditions described earlier (BME + 5-10% FCS). After 10 days in culture, less than 1% of N R cells are able to synthesize DNA, as shown by thymidine incorporation. Similar results on the kinetics of growth arrest in cultured N R ceils are obtained by studying incorporation of BrdU in replicating D N A (see Methods below). At days 1 and 2, after plating, the great majority of cells are in the S phase, whereas at day 5 only a few cells are labeled (Figs. 7A and 7B). N R cells can be maintained in culture for several weeks. Clusters of neuronal cells will disappear progressively; by day 21, most cells appear flattened. Eventually, all cells will degenerate. Changing culture conditions may influence the growth pattern of N R ceils. We have shown that these cells undergo about 10 divisions when cultured in Dulbecco's modified Eagle's medium ( D M E M ) supplemented with 10% FCS, 10% tryptose phosphate broth, and 2% heated chicken serum.l° The mechanisms by which specific components in different media may act on N R cell division are unknown and are worth studying. It would also be interesting to investigate potential effects of growth factors on NR cell division. E x p r e s s i o n of Retrovial O n c o g e n e s in NR Cells Infection of CNR and Q N R cultures with acutely transforming retroviruses, such as Rous sarcoma virus (RSV), results in transformation and sustained cell proliferation (Fig. 6B). An increase in the cell number becomes evident within 5-7 days. Dividing cells can be propagated during 20-30 generations, which represents a dramatic increase in their life span. However, RSV-infected CNR cells will eventually stop proliferating and undergo senescence. This process is accompanied by a progressive loss of the transformed phenotype, u I()C. B6chade and G. Calothy, Oncogene 6, 2311 (1991). it G. M. Seigel and M. F. D. Notter, J. Virol. 66, 6242 (1992).
FIG. 6. Morphology of normal and RSV-transformed NR cells. (A) Cultured ED7 QNR cells 3 days after plating. Note the presence of clusters of small differentiating neurons atop flat glial (Mtiller) cells. (B) RSV-transformed QNR cells 7 days after infection. Note the presence of typical round cells. These cells are larger than normal neurons. (C) Cultured ED7 QNR cells 5 weeks after plating showing the presence of typical lentoid bodies.
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FXG. 7. Detection of S phases with anti-BrdU antibodies in cultured cells. (A) Cultured ED7 QNR cells 2 days after plating. (B) Same culture after 5 days. (C) Clonal K2 cell line derived from ts NY68-infected QNR cells at 36 °. (D) K2 cells, made quiescent by transferring the cultures to 41.5° for 2 days.
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In RSV-infected cells, proliferation requires the expression of a functional p60 ..... as demonstrated by the existence of conditional mutants that are temperature sensitive (ts) for their mitogenic capacity.~2'13Proliferation of cells infected with such ts mutants can be, at will, switched on and off on their transfer to the appropriate temperature (Figs. 7C and 7D). Therefore, these cells are most suitable for dissecting mechanisms that lead to cell division. However, NR cells expressing an active p60 ...... also require the presence of growth factors present in the serum for their proliferation. in contrast to other cell types transformed by v-src. Such cells could be made quiescent by lowering the serum concentration to 0.1-0.5%, suggesting the possibility that two synergistic pathways are involved in the regulation of NR cell division. 14 NR cell proliferation can be measured either at the level of the cell population by cell counting, [3H]thymidine incorporation, 12or flow cytometry 14 or at the single cell level by determination of mitotic indexes by autoradiography11 or by BrdU labeling (see below). Results of flow cytometry analysis indicate that NR cells infected with ts mutants of RSV are arrested in G0/early G~ following inactivation of p60 ......a t a nonpermissive temperature. The S phase resumes synchronously within 12 hr following transfer to the permissive temperature. Similarly RSV-infected NR cells can be arrested in G1/GI, in the presence of 0.5% FCS at 370.14 Methods Infection with Retroviruses
Viral stocks should be filtered (0.22-/xm filters) to remove potential dividing cells. The addition of Polybrene (2-4/xg/ml) may increase virus uptake. CNR and QNR can be infected as cell suspensions, e.g., at the time of seeding, or as monolayers. The susceptibility of cultured NR cells to retroviral infection decreases as a function of time, in correlation with their withdrawal from the cell cycle. Similarly, NR cells dissected after ED10 are less responsive to viral infection. ~5 Infection of NR cells, dissected at ED6-8, with avian leukosis viruses (ALV) which do not induce NR cell proliferation yields virus titers that are comparable to those obtained in fibroblasts. Moreover, the virus production in proliferating NR cells infected 12G. Calothy, F. Poirier, G. Dambrine, P. Mignatti, P. Combes, and B. Pessac, Cold Spring Harbor Symp. Quant. Biol. 44, 983 (1980). 13F. Poirier, G. Calothy, R. E. Karess, E. Erikson, and H. Hanafusa, J. Virol. 42, 780 (1982). 14 G. Gillet, D. Michel, P. Crisanti, M. Gu6rin, Y. Herault, B. Pessac, G. Calothy, G. Brun. and M. Volovitch, Oncogene 8, 565 (1993). 15B. Pessac and G. Calothy, Science 185, 709 (1974).
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with acutely transforming viruses usually exceeds 107 infectious units (IU)/ ml. NR cells can also serve as host cells for nonreplicative retroviral vectors expressing selection markers and/or specific genes. DNA Transfer in NR Cells NR cells (5 x 106) (equivalent to one ED7 retina) are placed in a 35mm dish. The medium is changed the following day. Transfection is done 3 hr later as follows: 1. Prepare solutions A and B: Solution A: 250/zl 2x HBS and 5/zl 100x Phosphate. Solution B: 30/~1 2 M CaCI2, 5-30/zg DNA (5/zg of DNA is used for transfection of recombinant plasmids containing a replicative viral genome, whereas up to 30 /xg of DNA is required when transfecting recombinant DNA containing a nonreplicative genome), and up to 250/zl H20. 2. Add 250/xl of solution B dropwise to 250 /zl of solution A. The resulting precipitate is immediately added to the plate, without removing the medium. Five to twenty-four hr later, plates are washed with PBS, and fresh medium is added. Solutions 2× HBS: 10 g/liter HEPES and 16 g/liter NaC1, pH 7.1; sterilize by autoclaving or filtering. 100× Phosphate: 70 mM Na2HPO4 and 70 mM NaHzPO4; sterilize by autoclaving or filtering. 2 M CaC12, filter. H20, sterilize by autoclaving or filtering. Detection of S Phases with anti-BrdU Antibodies ON CULTUREDQNR CELLSINFECTEDWITHa ts MUTANTOF RSV. Cells are plated in complete medium on 2-cm2 dishes (2 x 104 cells/dish) and maintained at 36.5 ° for 5 days without medium renewal. At time t = 0, the culture medium is renewed and part of the cultures are shifted to 41.5 °. At each interval, BrdU is added to the medium, at a final concentration of 10 /xM, for 1 hr. Cells are incubated, fixed in 90% ethanol, and air dried. Slides are then immersed in 4 N HCI, neutralized in 0.1 M NazB407, pH 8.5, and treated with 50/xl of diluted mouse monoclonal anti-BrdU antibody (1/10 in PBS + 0.5% Tween 20) for 30 rain. After PBS washing, slides are incubated for 30 min with 50/zl of 0.5% Tween 20 in PBS containing a 1/20 dilution of fluorescein isothiocyanate (FITC)-conjugated F(ab')2
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goat anti-mouse, washed again in PBS, incubated for 1 min in 0.04 t~g/ml Evans blue (1/10,000) iodide, dried, and mounted for microscopic examination. ON EYE SECTIONS.Eyes are removed daily from incubated ED7-ED10 embryos. The iris is cut out and the vitrous humor is discarded, allowing contact between the NR and incubation medium. Eyes are incubated for 4 hr in complete medium containing 10/~M BrdU, fixed by immersion in paraformaldehyde (4% in PBS), and treated with 30% sucrose overnight. Eyes are frozen and sectioned at 10 ~m. Sections are dehydrated successively in 70, 95, and 100% ethanol and dried. BrdU incorporation is visualized as described earlier. Results of BrdU incorporation in ovo and in cultured cells are shown in Figs. 5 and 6. Establishment o f Clonal Cell Lines Derived f r o m Quail Retinas
We derived clonal lines from QNR cells transformed with a conditional mutant of RSV, tsNY68) 6 NR cells infected with this virus are transformed and proliferate at permissive temperatures (35-37°). They are morphologically normal and quiescent at nonpermissive temperatures (40-42°). Infected cells were first maintained at 39.5 ° for 4 weeks without subcultivation. At this intermediate temperature, QNR cells proliferate without being fully transformed. They were subsequently passaged three or four times over a period of 4 months after which the cells ceased to grow and underwent a "crisis" which lasted a few weeks. Cell growth then resumed and was maintained. Single cell derived clones were obtained by diluting the cultures according to Poisson distribution into 96-well plates in BME + 20% FCS. Cell suspensions were mixed with irradiated homologous cells, serving as feeder layers. Established clonal lines remained ts for cell proliferation (see Figs. 6C and 6D).
Use of NR Cells in Genetic and Functional Analysis of Oncogenes As mentioned earlier, the v-src gene is responsible for the mitogenic and transforming properties of RSV, as shown by the existence of v-src mutants that are ts for their capacity to induce NR cell division. However, these parameters are not always coordinately expressed in NR ceils. We previously reported that NR cells infected with certain ts mutants of RSV ~6B. Pessac,A. Girard,G. Romey,P. Crisanti.A. M.Lorinet,and G. Calothy,Nature (London) 302, 616 (1983).
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were induced to proliferate, at a nonpermissive temperature, in the absence of morphological transformation. 12J7 Based on this observation, we used N R cell proliferation as a phenotypic m a r k e r to select for mutants expressing phenotypic dissociation of mitogenic and transforming properties at permissive temperatures (35-37°). Two such mutants (PAl01 and P A l 0 4 ) were extensively characterized. 12'm8-2° Interestingly, both viruses exhibit very low tyrosine kinase activity] 3'2° suggesting that there was no direct correlation between the levels of tyrosine kinase activity and the ability to induce cell division. Molecular and functional analysis of these mutants showed the importance of the kinase domain in controlling all aspects of transformation. T h e y also established the importance of the amino-terminal protein of p60 v-src in modulating transformation p a r a m e t e r s and interacting with the kinase domain. It was proposed that the N-terminal portion of p60 vsr¢ could be involved in cell substrate recognition, a hypothesis widely confirmed since through the molecular and functional dissection of specific subdomains of p60 ..... (SH3 and SHe domains). Along these lines, we also showed that mutations that prevent myristoylation and binding of p60 v-sr¢ to the m e m brane do not abolish its mitogenic property. 21 T a k e n together, these results are consistent with the possibility that interaction of p60 ..... with a subset of cellular substrates leads to changes in growth regulation of N R cells. In another set of experiments, N R cells were used to analyze the functions of avian retrovirus M H 2 which carries both the m i l / r a f and the m y c oncogene. Infection of N R cells with this virus results in morphological transformation and sustained proliferation. Isolation of N R cells of spontaneous M H 2 mutants bearing a deletion of the v - m y c gene showed that vm i l / r a f was sufficient to induce N R cell proliferation. 22 I n d u c t i o n of NR Cell Proliferation b y A v i a n L e u k o s i s Viruses: A Model for S t u d y i n g O n c o g e n e T r a n s d u c t i o n b y R e t r o v i r u s e s We reported that avian leukosis viruses, which do not carry an oncogene, also induce N R cell proliferation. However, cell division is observed in a 17G. Calothy and B. Pessac, Virology 71, 336 (1976). 18B. J. Mayer, R. Jove, J. F. Krane, F. Poirier, G. Calothy, and H. Hanafusa, J. Virol. 60, 858 (1986). ~9R. Jove, B. J. Mayer, H. Iba, D. Laugier, F. Poirier, G. Calothy, T. Hanafusa, and H. Hanafusa, J. Virol. 60, 840 (1986). z~R. Jove, E. A. Garber, H. Iba, and H. Hanafusa, J. Virol. 60, 849 (1986). 21 G. Calothy, D. Laugier, F. R. Cross, R. Jove, T. Hanafusa, and H. Hanafusa, J. Virol. 61, 1678 (1987). 22C. B6chade, G. Calothy, B. Pessac, P. Martin, J. Coil, F. Denhez, S. Saule, J. Ghysdael, and D. St6h61in, Nature (London) 316, 559 (1985).
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small minority of cells after a long delay (several weeks), which contrasts with the massive and rapid proliferation caused by oncogene-containing retroviruses. This suggests that A L V activate, presumably by insertional mutagenesis, genes that are responsible for N R cell multiplication. By serially passaging supernatants of proliferating cells on fresh N R cultures, we reproducibly isolated new acutely mitogenic retroviruses that are capable of inducing N R cell division within a short delay. We showed that all retroviruses, thus far isolated, transduced the catalytic domain of two related serine/threonine protein k i n a s e s : c - m i l / c - r a f and c - R m i l / B - r a f 23"24 By analyzing retroviruses isolated at different passages on N R cells, we were able to establish that a crucial step in oncogene transduction is the formation of hybrid transcripts containing viral and activated cellular sequences (e.g., m U and R m i l ) . Such transcripts are presumably generated by occasional read-through transcription escaping termination signals in the 3' L T R 25 and are responsible for N R proliferation. > They are subsequently packaged and acquire 3' viral sequences by recombination with the viral genome z6 favored by partial sequence identity, eT'2s Characterization of such read-through transcripts in correlation with the acquisition of growth capacity in N R cells infected with A L V may prove useful in identifying genes involved in the regulation of growth or proliferation of these cells.
Analysis of Retina-Specific Differentially R e g u l a t e d G e n e s b y in S i t u H y b r i d i z a t i o n N R cells infected with conditional mutants of RSV represent a useful model to study mechanisms regulating the growth of differentiated cells. Our experimental approach in this direction was to isolate, by differential screening, cDNAs corresponding to m R N A s specifically expressed in nondividing N R cells (postmitotic N R cells in o v o and ceils made quiescent after shifting ts RSV-infected cultures to nonpermissive temperature) and down-regulated in proliferating ceils. We used in situ hybridization to study 23M. Marx, P. Crisanti, A. Eych6ne, C. B6chade, D. Laugier, J. Ghysdael, B. Pessac, and G. Calothy, J. Virol. 62, 4627 (1988). 24M. Marx, A. Eych6ne, D. Laugier, C. B6chade, P. Crisanti, P. Dez616e, B. Pessac, and G. Calothy, EMBO J. 7, 3369 (1988). 2s S. A. Herman and J. M. Coffin,J. Virol. 60, 497 (1986). 26M. P. Felder, D. Laugier, A. Eych~ne, G. Calothy, and M. Marx, J. Virol. 67, 6853 (1993). 27A. Eych6ne, C. Bdchade, M. Marx, D. Laugier, P. Dez616e, and G. Calothy, J. ViroL 64, 231 (1990). 2, M. P. Felder, A. Eych6ne, J. V. Barnier, I. Calogeraki, G. Calothy, and M. Marx, J. Virol. 65, 3633 (1991).
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the transcription of two retina-specific genes, QN129 (Fig. 8) and QR13° (Fig. 9), during development of the NR. Both genes encode presumably secreted proteins that could play a role in the regulation of cell division and/or cell to cell interactions.
Methods
Eyes of embryonic or postnatal quails were fixed overnight in 4% paraformaldehyde in PBS and then treated overnight in 30% sucrose. Sections of 15/xm were freeze-cut and hybridized as described with RNAs probes or oligonucleotides probes. R N A Probes
POLYMERASE. The D N A to be transcribed should be cloned into the polylinker site of an appropriate transcription vector which contains a promoter for SP6 T3 or T7 R N A polymerase. LABELING REACTION: Transcription buffer 10x concentrated: 400 mmol/liter, Tris-HC1, pH 8 (20°), 60 mmol/liter MgC12, 100 mmol/liter disthiothreitol, 20 mmol/liter, spermidine, 100 mmol/liter NaC1, and 1 U//xl RNase inhibitor. 1. The linearized D N A to be transcribed should be purified by phenol/ chloroform extraction and ethanol precipitation. 2. Add the following mixture to a microfuge tube on ice: 1/xg of linearized D N A 2/xl DIG NTP labeling mixture, 10x concentration 2 /zl 10x transcription buffer made up to 18 /xl with sterile water and add 2/xl SP6 (40 U) T7 or T3 R N A polymerase. 3. Centrifuge briefly and incubate for 2 hr at 37°. A longer incubation does not increase the yield of labeled RNA. 4. As the amount of the DIG-labeled R N A transcript is far in excess of the template DNA, it is not usually necessary to remove the template D N A by DNase treatment. If desired, the template D N A can be removed by the direct addition of 20 U DNase I, RNase-free and incubation for 15 min at 37 °. 5. With or without prior DNase, add 2/xl 0.2 M EDTA solution, pH 8, to stop the reaction. 29 L. Bidou, P. Crisanti, C. Blancher, and B. Pessac, Mech. Dev. 43, 159 (1993). 30 M. Guermah, P. Crisanti, D. Laugier, P. Dez616e, L. Bidou, B. Pessac, and G. Calothy, Proc. Natl. Acad. Sci. U.S.A. 88, 4503 (1991).
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FIG. 8. Study of QN1 RNA expression during NR development by in situ hybridization. QN1 expression is compared to S phase detection (left) QN1 RNA is detected only in postmitotic neurons (right).
6. Precipitate the labeled R N A with 2.5/xl LiC1, 4 mol/liter and 75/xl prechilled (20°C) ethanol, mix well. 7. Leave for at least 30 min at - 7 0 ° or for 2 hr at - 2 0 °. 8. Centrifuge (at 12,000 g), wash the pellet with 50/xl cold ethanol, 70% (v/v), dry under vacuum, and dissolve for 30 min at 37 ° in 100 txl
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Flo. 9. Regulation of QR1 expression. (Top) Hybridization with sense (1) and antisense (2) RNA probes. QR1 is expressed in the middle of the inner nuclear layer. (Bottom) Northern blot analysis of QR1 transcription. (A) QR1 expression correlates with quiescence of NR cells infected with ts mutants of RSV. (B) QR1 is a retina-specific gene. (C) QR1 is regulated during NR development.
diethyl pryocarbonate-treated water; 20 U R N a s e inhibitor can be added to inhibit possible contaminating RNases. 9. The amount of newly synthesized labeled R N A depends on the amount, size (site of linearization), and purity of the template D N A . 10. The transcript can be analyzed by agarose gel electrophoresis and ethidium bromide staining; the yield of transcript can be estimated by comparing the ratio between D N A and R N A bands.
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Oligonucleotide Probes Oligonucleotides of 23- and 31-mer are tailed with DIG-dUTP as described in the Boehringer Mannheim kit. Hybridizations of retina sections were performed overnight at 37° with 10/zl of probes at a concentration of 10 to 20 ng//zl in 50% formamide, 5 x SSC, 0.02% SDS, DNA salmon sperm or 5% blocking reagent and 0.1% N-laurylsarcosine. After hybridization, sections were washed at room temperature in 2 x SSC for 30 min, in 1 x SSC for 30 min, and in 0.5 x SSC for 30 min; rapidly soaked in 100 mM Tris-HC1, 100 mM NaC1, pH 7.5 (buffer 1); and incubated in the 1% (w/v) blocking reagent (w:v) from Boehringer Mannheim (buffer 2) or with 50/~g/ml of D N A salmon sperm. Polyclonal sheep antidigoxigenin Fab fragments conjugated to alkaline phosphatase (750 U/mg) were diluted with buffer 1 and applied to sections for 30 min at room temperature. Slides were washed with buffer 1 at room temperature and equilibrated with 100 mM Tris-HC1, 100 mM NaC1, and 50 mM MgC12, pH 9.5 (buffer 3); finally, slides were incubated with freshly prepared NBT and X phosphate solution (45/zl of NBT at 70 mg/ml and 35/zl of X phosphate at 50 mg/ml) in 10 ml of buffer 3. This incubation was in a humidified light-tight box (in the dark) without shaking for 3 hr or more. Reactions were stopped with 10 mM Tris-HC1, 1 mM EDTA, pH 8. Conclusion NR cells proved useful in oncogene studies in several aspects. They allowed to establish model cell systems in which growth and differentiation are controlled by oncogene expression. They also provided a tool for genetic and functional analysis of oncogenes by showing that cell proliferation induced by oncogenes could be dissociated from the expression of other transformation markers. Finally, they provided a convenient model for in vitro studies on activation and transduction of protooncogenes by retroviruses. Acknowledgments We gratefully acknowledge support from the Association pour la Recherche sur le Cancer and the Association Fran~aise Retinitis Pigmentosa.
AVIAN NEURORETINA CELLS
[5] A v i a n N e u r o r e t i n a
By
77
Cells in Oncogene
PATRICIA CRISANTI, BERNARD PESSAC,
and
Studies
GEORGES CALOTHY
There are two main reasons to use cells from the avian embryonic neuroretina (NR) as an experimental system to study the effects of oncogenes on regulation of cell growth and differentiation. First, NR cultures are exclusively composed of neuroectodermal cells: this tissue is not vascularized and does not contain cells of mesenchymal origin. Second, when these cells are maintained in culture, they rapidly stop dividing and express several markers of neural dfferentiation. The NR is part of the central nervous system and is, therefore, of ectodermal origin. It is derived from the optic cup which is an evagination from the wall of the diencephalon. The optic cup consists of an external layer which gives rise to the pigment epithelium and an internal layer which develops into the NR itself (Fig. 1). 12 A schematic representation of the vertebrate NR is shown in Fig. 2. The adult NR is composed of three types of neurons, photoreceptors, bipolar cells, and ganglion cells, and of two types of interneurons, horizontal and amacrine cells and one type of glial cells (MUller cells). These cells are organized in a layered structure together with two synaptic layers: the outer and the inner plexiform layers (Fig. 2). Two main types of synaptic complexes are present in the NR: the nonribbon (conventional synapses that are found in all neural tissues) and the ribbon synapses that are specific of the NR (Fig. 3). 3 Neuroretina Development and Differentiation The avian NR development proceeds through essentially three phases: proliferation of neuroectodermal precursor ceils, lamination of cell strata, and differentiation of postmitotic cells. 4 During early phases of development, immature cells are present across the entire primitive NR, and mitotic cells are visible in the external part adjacent to the future pigment epithelium. At a precise stage of development, cells become postmitotic and migrate toward the inner face of the NR. They then start to differentiate i R. Adler and D. Farber, Eds. " T h e Retina, Part 1: A Model for Cell Biology Studies." Academic Press, Orlando, FL, 1986. 2 N. J. Berrill and G. Karp, " D e v e l o p m e n t . " McGraw-Hill, New York, 1976. P. Crisanti-Combes, A., Privat, B. Pessac, and G. Calothy, Cell Tissue Rex. 185, 159 (1977). 4 C. J. Barnstable, Mol. Neurobiol. 1, 9 (1987).
METHODS IN ENZYMOLOGY.VOL. 254
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Fie. 2. Synaptic contacts between the main types of retinal cells. The upper layer consists of rods and cones. These sensory cells make synaptic contact with their neighboring cells and with bipolar and horizontal cells. These in turn make synaptic contacts with the inner ganglion cells whose axons constitute the optic nerve (from Berrill and Karfa).
when reaching positions that define their corresponding layers. Thymidine incorporation experiments, at various stages of chicken embryonic development, show that withdrawal from the cell cycle follows a posterior --~ anterior gradient: cells from the posterior pole, close to the optic nerve, are the first to withdraw from the cell cycle and to differentiate. Moreover, all cells located within a given region do not exit the cycle synchronously. Precursors of ganglion cells are the first to stop dividing between ED3 (Day 3 of embryonic development) and EDS, followed by precursors of cones and rods which in contrast will differentiate shortly before hatching. At later stages, most horizontal and amacrine cells become postmitotic. Precursors of bipolar and Mfiller cells are the last to exit from cell cycle. Finally, at ED10, the vast majority of chicken NR cells are postmitotic. Synaptogenesis begins between ED10 and E D l l . Figure 4 describes representative steps of NR histogenesis in chicken embryos between ED6 and 1 day after hatching.
FIG. 3. (A) Typical diads and triads arrangement of synaptic ribbons associated with aligned synaptic vesicles. (B) A large presynaptic with a dense matrix containing asynaptic ribbons with vesicles. Identical synaptic junctions are observed in cultured NR cells.
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Only a few reports have described the kinetics of cell cycle withdrawal in embryonic quail NR (QNR) cells. 5 Because the development of the quail proceeds at a faster rate than that of the chicken, both withdrawal from the cell cycle and lamination take place earlier (17 days vs 20 days in the chicken). Investigation of the kinetics and topography of D N A arrest in the embryonic QNR, by bromodeoxyuridine (BrdU) incorporation in replicating D N A (see methods below), led to the following observations (Fig. 5): At ED7 (Fig. 5A), the lamination process has just begun. Only the ganglion cells are organized in a distinct lamina which is not yet separated from the other cells by the plexiform layer. The remaining NR appears as a homogeneous population of elongated cells in the process of migration. At this stage, no labeled nuclei are observed in ganglion cells whereas a number of fluorescent nuclei are homogenously distributed over a wide zone of inorganized cells. At ED9 (Fig. 5B), cells of the presumptive inner nuclear layer (INL) are still elongated and intermingled. Only the inner plexiform layer (IPL) starts to be individualized. The number of S phases is markedly reduced in comparison to ED7 and ED8. Labeled nuclei are localized in the presumptive INL. The posterior pole appears more differentiated and presents a reduced number of S phases as compared to the anterior pole. At ED10 (Fig. 5C), the lamination process is not achieved but the IPL is visible, while the outer segments of photoreceptors are in formation. At this stage, no labeled nuclei are visible in either pole. Cells could not be labeled with BrdU thereafter.
Dissection NR can be dissected from chicken (CNR) or quail (QNR) embryos as early as days 6-7 of incubation. 3 Heads are first immersed in phosphatebuffered saline (PBS) without Cae+/Mg 2÷ (PBS-) and are parted in two along the median axis with fine forceps. Eyes are dissected out, the lenses are discarded, and the NR is carefully separated from the underlying pigment epithelium under a dissecting microscope. The retinas are spread in PBSand the few adhering pieces of pigment epithelium are cut away. Retinas are pooled, washed twice in PBS-, incubated in PBS- for 10 min at 37 °, and then in a 0.25% (w/v) trypsin solution in PBS- for 20 min. After centrifugation at room temperature, NR are washed three times in Eagle's 5 A. A. Moscona and L. Degenstein, Dev. Neurosci. 4, 211 (1981).
.......
7
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FIG. 5. Detection of S phases during various stages of development: (A) at ED7, (B) ED9, and (C) ED10. Incorporation of BrdU into replicating D N A was detected by indirect immunofluorescence. See text.
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basal medium (BME), without dissociation, and finally, tissues are dissociated by gentle pipetting in BME supplemented with 5-10% fetal calf serum (FCS) and antibiotics (100/zg/ml kanamycin, 100/zg/ml streptomycin, and 100 units/ml penicillin). The cells are centrifuged at 200 g, resuspensed in culture medium, and counted in a hemocytometer. They appear as single cells with a few tiny clumps. One ED7 NR yields about 5 x 106 cells. They are usually seeded at a density of 3 x 105 cells/cm2 and are incubated at 37 ° in a humidified atmosphere [5% (v/v) CO2 in air]. Medium is renewed every second or third day.
Light and Electron Microscopy Studies of NR Cultures Cultures are observed by light microscopy or processed for electron microscopy as follows: they are fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 hr and postfixed in 1% osmium tetroxide in the same buffer for 1 hr. Fixed cells are stained en bloc with uranyl acetate, dehydrated in ethanol-acetate, and fiat-embedded in Araldite. Ultrathin sections are cut in a plane parallel to the substrate. NR dissected from embryos at the onset of the cultures are processed similarly for electron microscopy. After 24 hr, NR cells from 6 to 7-day-old chicken embryos are composed of clusters of small, round cells (seven to eight times smaller than fibroblasts) that are undifferentiated neuroblasts. Two to 3 days later, these small clusters spread out and start to extend processes. Round cells differentiate from very immature cell types, neuroepithelial like at the time of dissociation, into relatively mature neurons. This maturation process is visualized by the development of perikaryal organelles and transformation of the nucleus from a fusiform notched outline, with numerous chromatine clumps, to a regular, spherical shape with an evenly dispersed chromatin and a prominent nucleolus. In addition, processes grow out of these cells, progress with growth cones on the flattened cells, and form a reticular array between groups of round cells. However, it is difficult, if not impossible, to identify most of these neurons. Photoreceptors cells are identifiable because of the presence of ribbon synapses characteristic of cone receptors in the adult retina. Similarly, ganglion cells could be tentatively identified because of their size, larger than that of any other neuron in culture, and of their general morphology, especially because of the presence of an axon. Areas of neuropiles are distributed in the vicinity of neuronal clusters and, sometimes, in the center of such clusters. They more or less mimic the organization of the retina into cellular and plexiform layers. Synapses are found after 8 days in vitro and are still visible after 15
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and 21 days in culture. They correspond to the different types found in the retina in vivo, although they most often lack the typical organization of postsynaptic elements. A second cell type becomes morphologically distinguishable and is composed of fiat, transparent cells that may represent MOiler cells for two reasons. First, they do not show neuronal characteristics. Second, they display some ultrastructural similarities to MOiler cells of the in vivo retina, namely the presence of large arrays of filaments and microtubules, as well as numerous and extensive attachment plates associated with some of the fibrillar arrays (Fig. 6A). In summary, NR cultures obtained from 7-day-old chicken embryos can be maintained as monolayers and are able to differentiate into neuronal and nonneuronal (glial) cells. This differentiation proceeds to the stage at which morphologically mature synapses are found. It, therefore, appears that synaptogenesis primarily depends on the genetic programs of the cells and that complex interactions, found in tridimensional structures, may not be required for the formation of synapses.
Transdifferentiation From the fourth week onward, new phenotypes appear that might be due to transdifferentiation6 of cells that were initially programmed into the neuronal or glial pathways or otherwise derived from uncommitted stem cells. One of the phenotypes is made of small pigmented cells, whereas the second consists of cells that progressively take on the appearance of lentoid bodies (LB). These structures are formed of densely packed refringent cells and of "bottle cells," both of which appear in culture in lens epithelial cells and follow a differentiation pathway similar to that of the lens (Fig. 6C). The LB are always associated with crystallins identical to those present in lens during development or in cultured lens epithelial cells. Crystallins constitute the bulk of lens proteins and, in birds, fall into three major classes (c~,/3, and 8) that are the products of three multigene families.7,~ c~and 8-crystallins are present on day 28 in cultured QNR and progressively increase until day 42. The amounts of a- and 6-crystallins remain stable thereafter. In QNR cultures passaged once after 10 weeks, the levels of c~and 8-crystallins remain elevated. 9
T. S. Okada, Y. Itoh, K. Watanabe, and G. Eguchi, Dev. Biol. 45, 318 (1975). v D. I. De Pomerai, D. J. Pritchard, and R. M. Clayton, Dev. Biol. 60, 416 (1977). 8 R. M. Clayton, I. Thompson, and D. I. De Pomerai, Nature (London) 282, 628 (1979). 9 L. Simonneau, P. Crisanti, A. M. Lorinet, F. Alliot, Y. Courtois, G. Calothy, and B. Pessac, Mol. CelL Biol. 6, 3704 (1986).
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[5]
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W i t h d r a w a l from Cell Cycle of C u l t u r e d NR Cultures of CNR or Q N R ceils dissected at E D 6 - E D 7 undergo two or three doublings at the level of cell population under the culture conditions described earlier (BME + 5-10% FCS). After 10 days in culture, less than 1% of N R cells are able to synthesize DNA, as shown by thymidine incorporation. Similar results on the kinetics of growth arrest in cultured N R ceils are obtained by studying incorporation of BrdU in replicating D N A (see Methods below). At days 1 and 2, after plating, the great majority of cells are in the S phase, whereas at day 5 only a few cells are labeled (Figs. 7A and 7B). N R cells can be maintained in culture for several weeks. Clusters of neuronal cells will disappear progressively; by day 21, most cells appear flattened. Eventually, all cells will degenerate. Changing culture conditions may influence the growth pattern of N R ceils. We have shown that these cells undergo about 10 divisions when cultured in Dulbecco's modified Eagle's medium ( D M E M ) supplemented with 10% FCS, 10% tryptose phosphate broth, and 2% heated chicken serum.l° The mechanisms by which specific components in different media may act on N R cell division are unknown and are worth studying. It would also be interesting to investigate potential effects of growth factors on NR cell division. E x p r e s s i o n of Retrovial O n c o g e n e s in NR Cells Infection of CNR and Q N R cultures with acutely transforming retroviruses, such as Rous sarcoma virus (RSV), results in transformation and sustained cell proliferation (Fig. 6B). An increase in the cell number becomes evident within 5-7 days. Dividing cells can be propagated during 20-30 generations, which represents a dramatic increase in their life span. However, RSV-infected CNR cells will eventually stop proliferating and undergo senescence. This process is accompanied by a progressive loss of the transformed phenotype, u I()C. B6chade and G. Calothy, Oncogene 6, 2311 (1991). it G. M. Seigel and M. F. D. Notter, J. Virol. 66, 6242 (1992).
FIG. 6. Morphology of normal and RSV-transformed NR cells. (A) Cultured ED7 QNR cells 3 days after plating. Note the presence of clusters of small differentiating neurons atop flat glial (Mtiller) cells. (B) RSV-transformed QNR cells 7 days after infection. Note the presence of typical round cells. These cells are larger than normal neurons. (C) Cultured ED7 QNR cells 5 weeks after plating showing the presence of typical lentoid bodies.
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FXG. 7. Detection of S phases with anti-BrdU antibodies in cultured cells. (A) Cultured ED7 QNR cells 2 days after plating. (B) Same culture after 5 days. (C) Clonal K2 cell line derived from ts NY68-infected QNR cells at 36 °. (D) K2 cells, made quiescent by transferring the cultures to 41.5° for 2 days.
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In RSV-infected cells, proliferation requires the expression of a functional p60 ..... as demonstrated by the existence of conditional mutants that are temperature sensitive (ts) for their mitogenic capacity.~2'13Proliferation of cells infected with such ts mutants can be, at will, switched on and off on their transfer to the appropriate temperature (Figs. 7C and 7D). Therefore, these cells are most suitable for dissecting mechanisms that lead to cell division. However, NR cells expressing an active p60 ...... also require the presence of growth factors present in the serum for their proliferation. in contrast to other cell types transformed by v-src. Such cells could be made quiescent by lowering the serum concentration to 0.1-0.5%, suggesting the possibility that two synergistic pathways are involved in the regulation of NR cell division. 14 NR cell proliferation can be measured either at the level of the cell population by cell counting, [3H]thymidine incorporation, 12or flow cytometry 14 or at the single cell level by determination of mitotic indexes by autoradiography11 or by BrdU labeling (see below). Results of flow cytometry analysis indicate that NR cells infected with ts mutants of RSV are arrested in G0/early G~ following inactivation of p60 ......a t a nonpermissive temperature. The S phase resumes synchronously within 12 hr following transfer to the permissive temperature. Similarly RSV-infected NR cells can be arrested in G1/GI, in the presence of 0.5% FCS at 370.14 Methods Infection with Retroviruses
Viral stocks should be filtered (0.22-/xm filters) to remove potential dividing cells. The addition of Polybrene (2-4/xg/ml) may increase virus uptake. CNR and QNR can be infected as cell suspensions, e.g., at the time of seeding, or as monolayers. The susceptibility of cultured NR cells to retroviral infection decreases as a function of time, in correlation with their withdrawal from the cell cycle. Similarly, NR cells dissected after ED10 are less responsive to viral infection. ~5 Infection of NR cells, dissected at ED6-8, with avian leukosis viruses (ALV) which do not induce NR cell proliferation yields virus titers that are comparable to those obtained in fibroblasts. Moreover, the virus production in proliferating NR cells infected 12G. Calothy, F. Poirier, G. Dambrine, P. Mignatti, P. Combes, and B. Pessac, Cold Spring Harbor Symp. Quant. Biol. 44, 983 (1980). 13F. Poirier, G. Calothy, R. E. Karess, E. Erikson, and H. Hanafusa, J. Virol. 42, 780 (1982). 14 G. Gillet, D. Michel, P. Crisanti, M. Gu6rin, Y. Herault, B. Pessac, G. Calothy, G. Brun. and M. Volovitch, Oncogene 8, 565 (1993). 15B. Pessac and G. Calothy, Science 185, 709 (1974).
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with acutely transforming viruses usually exceeds 107 infectious units (IU)/ ml. NR cells can also serve as host cells for nonreplicative retroviral vectors expressing selection markers and/or specific genes. DNA Transfer in NR Cells NR cells (5 x 106) (equivalent to one ED7 retina) are placed in a 35mm dish. The medium is changed the following day. Transfection is done 3 hr later as follows: 1. Prepare solutions A and B: Solution A: 250/zl 2x HBS and 5/zl 100x Phosphate. Solution B: 30/~1 2 M CaCI2, 5-30/zg DNA (5/zg of DNA is used for transfection of recombinant plasmids containing a replicative viral genome, whereas up to 30 /xg of DNA is required when transfecting recombinant DNA containing a nonreplicative genome), and up to 250/zl H20. 2. Add 250/xl of solution B dropwise to 250 /zl of solution A. The resulting precipitate is immediately added to the plate, without removing the medium. Five to twenty-four hr later, plates are washed with PBS, and fresh medium is added. Solutions 2× HBS: 10 g/liter HEPES and 16 g/liter NaC1, pH 7.1; sterilize by autoclaving or filtering. 100× Phosphate: 70 mM Na2HPO4 and 70 mM NaHzPO4; sterilize by autoclaving or filtering. 2 M CaC12, filter. H20, sterilize by autoclaving or filtering. Detection of S Phases with anti-BrdU Antibodies ON CULTUREDQNR CELLSINFECTEDWITHa ts MUTANTOF RSV. Cells are plated in complete medium on 2-cm2 dishes (2 x 104 cells/dish) and maintained at 36.5 ° for 5 days without medium renewal. At time t = 0, the culture medium is renewed and part of the cultures are shifted to 41.5 °. At each interval, BrdU is added to the medium, at a final concentration of 10 /xM, for 1 hr. Cells are incubated, fixed in 90% ethanol, and air dried. Slides are then immersed in 4 N HCI, neutralized in 0.1 M NazB407, pH 8.5, and treated with 50/xl of diluted mouse monoclonal anti-BrdU antibody (1/10 in PBS + 0.5% Tween 20) for 30 rain. After PBS washing, slides are incubated for 30 min with 50/zl of 0.5% Tween 20 in PBS containing a 1/20 dilution of fluorescein isothiocyanate (FITC)-conjugated F(ab')2
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goat anti-mouse, washed again in PBS, incubated for 1 min in 0.04 t~g/ml Evans blue (1/10,000) iodide, dried, and mounted for microscopic examination. ON EYE SECTIONS.Eyes are removed daily from incubated ED7-ED10 embryos. The iris is cut out and the vitrous humor is discarded, allowing contact between the NR and incubation medium. Eyes are incubated for 4 hr in complete medium containing 10/~M BrdU, fixed by immersion in paraformaldehyde (4% in PBS), and treated with 30% sucrose overnight. Eyes are frozen and sectioned at 10 ~m. Sections are dehydrated successively in 70, 95, and 100% ethanol and dried. BrdU incorporation is visualized as described earlier. Results of BrdU incorporation in ovo and in cultured cells are shown in Figs. 5 and 6. Establishment o f Clonal Cell Lines Derived f r o m Quail Retinas
We derived clonal lines from QNR cells transformed with a conditional mutant of RSV, tsNY68) 6 NR cells infected with this virus are transformed and proliferate at permissive temperatures (35-37°). They are morphologically normal and quiescent at nonpermissive temperatures (40-42°). Infected cells were first maintained at 39.5 ° for 4 weeks without subcultivation. At this intermediate temperature, QNR cells proliferate without being fully transformed. They were subsequently passaged three or four times over a period of 4 months after which the cells ceased to grow and underwent a "crisis" which lasted a few weeks. Cell growth then resumed and was maintained. Single cell derived clones were obtained by diluting the cultures according to Poisson distribution into 96-well plates in BME + 20% FCS. Cell suspensions were mixed with irradiated homologous cells, serving as feeder layers. Established clonal lines remained ts for cell proliferation (see Figs. 6C and 6D).
Use of NR Cells in Genetic and Functional Analysis of Oncogenes As mentioned earlier, the v-src gene is responsible for the mitogenic and transforming properties of RSV, as shown by the existence of v-src mutants that are ts for their capacity to induce NR cell division. However, these parameters are not always coordinately expressed in NR ceils. We previously reported that NR cells infected with certain ts mutants of RSV ~6B. Pessac,A. Girard,G. Romey,P. Crisanti.A. M.Lorinet,and G. Calothy,Nature (London) 302, 616 (1983).
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were induced to proliferate, at a nonpermissive temperature, in the absence of morphological transformation. 12J7 Based on this observation, we used N R cell proliferation as a phenotypic m a r k e r to select for mutants expressing phenotypic dissociation of mitogenic and transforming properties at permissive temperatures (35-37°). Two such mutants (PAl01 and P A l 0 4 ) were extensively characterized. 12'm8-2° Interestingly, both viruses exhibit very low tyrosine kinase activity] 3'2° suggesting that there was no direct correlation between the levels of tyrosine kinase activity and the ability to induce cell division. Molecular and functional analysis of these mutants showed the importance of the kinase domain in controlling all aspects of transformation. T h e y also established the importance of the amino-terminal protein of p60 v-src in modulating transformation p a r a m e t e r s and interacting with the kinase domain. It was proposed that the N-terminal portion of p60 vsr¢ could be involved in cell substrate recognition, a hypothesis widely confirmed since through the molecular and functional dissection of specific subdomains of p60 ..... (SH3 and SHe domains). Along these lines, we also showed that mutations that prevent myristoylation and binding of p60 v-sr¢ to the m e m brane do not abolish its mitogenic property. 21 T a k e n together, these results are consistent with the possibility that interaction of p60 ..... with a subset of cellular substrates leads to changes in growth regulation of N R cells. In another set of experiments, N R cells were used to analyze the functions of avian retrovirus M H 2 which carries both the m i l / r a f and the m y c oncogene. Infection of N R cells with this virus results in morphological transformation and sustained proliferation. Isolation of N R cells of spontaneous M H 2 mutants bearing a deletion of the v - m y c gene showed that vm i l / r a f was sufficient to induce N R cell proliferation. 22 I n d u c t i o n of NR Cell Proliferation b y A v i a n L e u k o s i s Viruses: A Model for S t u d y i n g O n c o g e n e T r a n s d u c t i o n b y R e t r o v i r u s e s We reported that avian leukosis viruses, which do not carry an oncogene, also induce N R cell proliferation. However, cell division is observed in a 17G. Calothy and B. Pessac, Virology 71, 336 (1976). 18B. J. Mayer, R. Jove, J. F. Krane, F. Poirier, G. Calothy, and H. Hanafusa, J. Virol. 60, 858 (1986). ~9R. Jove, B. J. Mayer, H. Iba, D. Laugier, F. Poirier, G. Calothy, T. Hanafusa, and H. Hanafusa, J. Virol. 60, 840 (1986). z~R. Jove, E. A. Garber, H. Iba, and H. Hanafusa, J. Virol. 60, 849 (1986). 21 G. Calothy, D. Laugier, F. R. Cross, R. Jove, T. Hanafusa, and H. Hanafusa, J. Virol. 61, 1678 (1987). 22C. B6chade, G. Calothy, B. Pessac, P. Martin, J. Coil, F. Denhez, S. Saule, J. Ghysdael, and D. St6h61in, Nature (London) 316, 559 (1985).
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small minority of cells after a long delay (several weeks), which contrasts with the massive and rapid proliferation caused by oncogene-containing retroviruses. This suggests that A L V activate, presumably by insertional mutagenesis, genes that are responsible for N R cell multiplication. By serially passaging supernatants of proliferating cells on fresh N R cultures, we reproducibly isolated new acutely mitogenic retroviruses that are capable of inducing N R cell division within a short delay. We showed that all retroviruses, thus far isolated, transduced the catalytic domain of two related serine/threonine protein k i n a s e s : c - m i l / c - r a f and c - R m i l / B - r a f 23"24 By analyzing retroviruses isolated at different passages on N R cells, we were able to establish that a crucial step in oncogene transduction is the formation of hybrid transcripts containing viral and activated cellular sequences (e.g., m U and R m i l ) . Such transcripts are presumably generated by occasional read-through transcription escaping termination signals in the 3' L T R 25 and are responsible for N R proliferation. > They are subsequently packaged and acquire 3' viral sequences by recombination with the viral genome z6 favored by partial sequence identity, eT'2s Characterization of such read-through transcripts in correlation with the acquisition of growth capacity in N R cells infected with A L V may prove useful in identifying genes involved in the regulation of growth or proliferation of these cells.
Analysis of Retina-Specific Differentially R e g u l a t e d G e n e s b y in S i t u H y b r i d i z a t i o n N R cells infected with conditional mutants of RSV represent a useful model to study mechanisms regulating the growth of differentiated cells. Our experimental approach in this direction was to isolate, by differential screening, cDNAs corresponding to m R N A s specifically expressed in nondividing N R cells (postmitotic N R cells in o v o and ceils made quiescent after shifting ts RSV-infected cultures to nonpermissive temperature) and down-regulated in proliferating ceils. We used in situ hybridization to study 23M. Marx, P. Crisanti, A. Eych6ne, C. B6chade, D. Laugier, J. Ghysdael, B. Pessac, and G. Calothy, J. Virol. 62, 4627 (1988). 24M. Marx, A. Eych6ne, D. Laugier, C. B6chade, P. Crisanti, P. Dez616e, B. Pessac, and G. Calothy, EMBO J. 7, 3369 (1988). 2s S. A. Herman and J. M. Coffin,J. Virol. 60, 497 (1986). 26M. P. Felder, D. Laugier, A. Eych~ne, G. Calothy, and M. Marx, J. Virol. 67, 6853 (1993). 27A. Eych6ne, C. Bdchade, M. Marx, D. Laugier, P. Dez616e, and G. Calothy, J. ViroL 64, 231 (1990). 2, M. P. Felder, A. Eych6ne, J. V. Barnier, I. Calogeraki, G. Calothy, and M. Marx, J. Virol. 65, 3633 (1991).
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the transcription of two retina-specific genes, QN129 (Fig. 8) and QR13° (Fig. 9), during development of the NR. Both genes encode presumably secreted proteins that could play a role in the regulation of cell division and/or cell to cell interactions.
Methods
Eyes of embryonic or postnatal quails were fixed overnight in 4% paraformaldehyde in PBS and then treated overnight in 30% sucrose. Sections of 15/xm were freeze-cut and hybridized as described with RNAs probes or oligonucleotides probes. R N A Probes
POLYMERASE. The D N A to be transcribed should be cloned into the polylinker site of an appropriate transcription vector which contains a promoter for SP6 T3 or T7 R N A polymerase. LABELING REACTION: Transcription buffer 10x concentrated: 400 mmol/liter, Tris-HC1, pH 8 (20°), 60 mmol/liter MgC12, 100 mmol/liter disthiothreitol, 20 mmol/liter, spermidine, 100 mmol/liter NaC1, and 1 U//xl RNase inhibitor. 1. The linearized D N A to be transcribed should be purified by phenol/ chloroform extraction and ethanol precipitation. 2. Add the following mixture to a microfuge tube on ice: 1/xg of linearized D N A 2/xl DIG NTP labeling mixture, 10x concentration 2 /zl 10x transcription buffer made up to 18 /xl with sterile water and add 2/xl SP6 (40 U) T7 or T3 R N A polymerase. 3. Centrifuge briefly and incubate for 2 hr at 37°. A longer incubation does not increase the yield of labeled RNA. 4. As the amount of the DIG-labeled R N A transcript is far in excess of the template DNA, it is not usually necessary to remove the template D N A by DNase treatment. If desired, the template D N A can be removed by the direct addition of 20 U DNase I, RNase-free and incubation for 15 min at 37 °. 5. With or without prior DNase, add 2/xl 0.2 M EDTA solution, pH 8, to stop the reaction. 29 L. Bidou, P. Crisanti, C. Blancher, and B. Pessac, Mech. Dev. 43, 159 (1993). 30 M. Guermah, P. Crisanti, D. Laugier, P. Dez616e, L. Bidou, B. Pessac, and G. Calothy, Proc. Natl. Acad. Sci. U.S.A. 88, 4503 (1991).
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FIG. 8. Study of QN1 RNA expression during NR development by in situ hybridization. QN1 expression is compared to S phase detection (left) QN1 RNA is detected only in postmitotic neurons (right).
6. Precipitate the labeled R N A with 2.5/xl LiC1, 4 mol/liter and 75/xl prechilled (20°C) ethanol, mix well. 7. Leave for at least 30 min at - 7 0 ° or for 2 hr at - 2 0 °. 8. Centrifuge (at 12,000 g), wash the pellet with 50/xl cold ethanol, 70% (v/v), dry under vacuum, and dissolve for 30 min at 37 ° in 100 txl
96
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ONL ~-
INL
'P'
[_
GCL
A OOOOOO
ZZZZZZZ
OOOOOOO
B
C
H NRB L i L u H K F
7 8 9 101114+1+3
QR 13Kb -
GAPDH
Flo. 9. Regulation of QR1 expression. (Top) Hybridization with sense (1) and antisense (2) RNA probes. QR1 is expressed in the middle of the inner nuclear layer. (Bottom) Northern blot analysis of QR1 transcription. (A) QR1 expression correlates with quiescence of NR cells infected with ts mutants of RSV. (B) QR1 is a retina-specific gene. (C) QR1 is regulated during NR development.
diethyl pryocarbonate-treated water; 20 U R N a s e inhibitor can be added to inhibit possible contaminating RNases. 9. The amount of newly synthesized labeled R N A depends on the amount, size (site of linearization), and purity of the template D N A . 10. The transcript can be analyzed by agarose gel electrophoresis and ethidium bromide staining; the yield of transcript can be estimated by comparing the ratio between D N A and R N A bands.
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Oligonucleotide Probes Oligonucleotides of 23- and 31-mer are tailed with DIG-dUTP as described in the Boehringer Mannheim kit. Hybridizations of retina sections were performed overnight at 37° with 10/zl of probes at a concentration of 10 to 20 ng//zl in 50% formamide, 5 x SSC, 0.02% SDS, DNA salmon sperm or 5% blocking reagent and 0.1% N-laurylsarcosine. After hybridization, sections were washed at room temperature in 2 x SSC for 30 min, in 1 x SSC for 30 min, and in 0.5 x SSC for 30 min; rapidly soaked in 100 mM Tris-HC1, 100 mM NaC1, pH 7.5 (buffer 1); and incubated in the 1% (w/v) blocking reagent (w:v) from Boehringer Mannheim (buffer 2) or with 50/~g/ml of D N A salmon sperm. Polyclonal sheep antidigoxigenin Fab fragments conjugated to alkaline phosphatase (750 U/mg) were diluted with buffer 1 and applied to sections for 30 min at room temperature. Slides were washed with buffer 1 at room temperature and equilibrated with 100 mM Tris-HC1, 100 mM NaC1, and 50 mM MgC12, pH 9.5 (buffer 3); finally, slides were incubated with freshly prepared NBT and X phosphate solution (45/zl of NBT at 70 mg/ml and 35/zl of X phosphate at 50 mg/ml) in 10 ml of buffer 3. This incubation was in a humidified light-tight box (in the dark) without shaking for 3 hr or more. Reactions were stopped with 10 mM Tris-HC1, 1 mM EDTA, pH 8. Conclusion NR cells proved useful in oncogene studies in several aspects. They allowed to establish model cell systems in which growth and differentiation are controlled by oncogene expression. They also provided a tool for genetic and functional analysis of oncogenes by showing that cell proliferation induced by oncogenes could be dissociated from the expression of other transformation markers. Finally, they provided a convenient model for in vitro studies on activation and transduction of protooncogenes by retroviruses. Acknowledgments We gratefully acknowledge support from the Association pour la Recherche sur le Cancer and the Association Fran~aise Retinitis Pigmentosa.