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Involvement of L-DNase II in Nuclear Degeneration during Chick Retina Development
Exp. Eye Res. (2001) 72, 443±453 doi:10.1006/exer.2000.0969, available online at http://www.idealibrary.com on
Involvement of L-DNase II in Nuclear Degeneration during Chick Retina Development AL IC IA TORR IGL IA* , E L I S A B E T H C H A U D U N , F R A N CË O I S E C H A N Y - FO U R N I E R , Y V E S CO U R TO I S A N D M A R IE - F R A N C E CO U N I S Unite 450 INSERM, Af®lieÂe CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France (Received Milwaukee 21 June 2000, accepted in revised form 8 December 2000 and published electronically 8 February 2001) During the development of the neural retina, 50 % of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the ®nal event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2 -Mg2 -dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using puri®ed nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase # 2001 Academic Press is responsible for most of the DNA degradation observed in these cells. Key words: DNases; retina development; apoptosis; L-DNase II; DNase I.
1. Introduction The retina is derived from the central nervous system, from an invagination of the proencephalin, called the optical vesicle. In the early stages of embryonic development, the retina is composed of undifferentiated neuro-epithelial cells. As the embryonic development proceeds the neuro-epithelium differentiates into three layers (Cepko, 1984). The outer layer contains the sensorial cells, the photoreceptors; the inner nuclear layer is formed by the ganglion cells. Between these two layers, the nuclei of the intermediate neurons are found. These nuclear layers are separated by the so-called plexiform layers which contain the synaptic regions and the axonal extensions. During the onset of this differentiation, the neurons which fail to establish the right synaptic connections are eliminated. In chick embryo, the retinal organogenesis starts at E8 and is almost complete at E18. During this period almost 50 % of the cells die. This cellular death occurs by the mechanism of apoptosis (Young, 1984). Waves of apoptosis sweep through the ganglion cell layer, inner nuclear layer and ®nally the photoreceptor layer as each region undergoes differentiation (Ilschner and Warning, 1992; Prindull, 1995). Apoptosis was described by Wyllie (1980) as a unique form of cell death that could be distinguished from necrosis induced by toxins and anoxic injury. The morphological hallmarks include cytoplasmic and nuclear condensation and fragmentation of cells into membrane-bound compartments (apoptotic * Author for correspondence. E-mail: [email protected]
0014-4835/01/04044311 $35.00/0
bodies) (Leach, 1998). These apoptotic cells are phagocytozed by adjacent cells and occasionally by resident macrophages. According to the current concepts, apoptosis is triggered by diverse interacting signals including expression of oncogenes (e.g. bcl-2, c-myc) (Yang and Korsmeyer, 1996; Prendergast, 1999) and tumor suppressor genes (Choisy-Rossi and Yonish-Rouach, 1998; Kasten and Giordano, 1998). As the molecular mechanism of apoptosis begin to be elucidated, the morphological criteria have been replaced by biochemical indicators such as DNA fragmentation, visualized in agarose gel electrophoresis as the `oligonucleosomal ladder' (Collins et al., 1992; Walker and Sikorska, 1995). Nucleosomal laddering is now viewed as a late and inconstant sign of apoptosis (Collins et al., 1992). Earlier stages are evidenced by high molecular DNA fragments (Brown, Sun and Cohen, 1993; Oberhammer et al., 1993). This cleavage of the genomic DNA is performed by endonucleases. Many studies have suggested that this is a Ca2 -dependent process (Gaido and Cidlowski, 1991; Nicotera and Rossi, 1994), but Kluck et al. (1994) have shown several instances where apoptosis was not associated with elevated intracellular Ca2 levels. Barry and Eastman proposed that under certain conditions DNase II, a Ca2 -independent DNase, may be activated by intracellular acidi®cation (Barry and Eastman, 1992; Eastman, 1994). Recent studies performed in our laboratory, analysing terminal differentiation in lens cells, seem to con®rm this conclusion (Torriglia et al., 1995; Counis et al., 1998). We have recently characterized the DNase responsible for this degradation. # 2001 Academic Press
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This enzyme has a DNase II-like activity, i.e. it is Ca2 Mg2 -independent and presents an optimal activity at acidic pH. This DNase is derived by post-translational modi®cation of the leukocyte elastase inhibitor (LEI) and it is called L-DNase II (Torriglia et al., 1998). In this study we tried to identify the endonuclease activated during apoptosis in the developing retina. This is an important piece of knowledge to investigate the apoptotic pathways activated to eliminate useless cells. We have identi®ed Ca2 -Mg2 -dependent and acidic endonucleases, in the developing chick retina, by measuring the activity of both enzymes in the total and nuclear extracts and by using antibodies against DNase I and L-DNase II. We show that both DNase I and L-DNase II, as well as other Ca2 -Mg2 dependent DNases are activated. DNase I does not seem to play a major role, but the implication of L-DNase II and the activation of its speci®c pathway is discussed.
2. Materials and Methods Materials Fertilized eggs of white Leghorn chicken were obtained from Hass (Kalthenhouse, France). Calf thymus DNA was from Pharmacia. Puri®ed DNase I was from Sigma and L-DNase II was from Worthington. Deoxy [8-3H] adenosine 50 triphosphate, ammonium salt (code TRK 34, 777 GBq mmol ÿ1) and enhanced chemiluminescence kit (ECL) were from Amersham International. Immobilon P was from Millipore. Escherichia coli DNA polymerase I (endonuclease free), dCTP, dGTP, dTTP were from Boehringher Mannheim. Polyclonal antibody against DNase I was from Rockland and biotinylated goat anti-rabbit IgG were from Cooper Biomedica. The scintillation liquid was Luma Safe from Packard. Prestained molecular weight markers for electrophoresis were from BioRad. The mounting material, Tissue Tek OCT, was obtained from Miles. The polyclonal antibody directed against DNase I was from Rockland. DNA Labeling Calf thymus DNA (0.5 mg) was 3H-labeled in 10 ml 50 mM Tris±HCl, pH 7.4 containing 5 mM MgCl2 , 50 mg ml ÿ1 bovine serum albumin, 10 mM 2mercaptoethanol, 50 mM dCTP, dGTP and dTTP, 2.5 nM 3HdATP and 100 IU DNA polymerase I. The reaction was allowed to proceed for 30 min at 158C then stopped with the addition of NaCl to a concentration of 150 mM. The labeled DNA was then precipitated with two volumes of 100 % ethanol for 2 hr at 08C. The precipitated DNA was washed four times with 70 % ethanol and centrifuged each time at 2500 rpm for 10 min. After air drying of the pellet, the labeled DNA was solubilized at 48C in 1 ml
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H2O. The concentration of solubilized DNA was evaluated by spectrophotometric reading at 260 nm. Measurement of DNase Activities DNase I activity was measured in 200 ml 10 mM Tris±HCl pH 7.4 containing 10 mM CaCl2 and 10 mM MgCl2 . The reaction, started with 1 mg 3H-DNA, was allowed to proceed for 30 min at 378C, then stopped by incubation for 1 hr at 08C with 200 ml of 10 % trichloroacetic acid (TCA) containing 1 % sodium pyrophosphate (PPi). The nondigested DNA was then trapped on a GF/C ®lter (Millipore) previously soaked in 1 % PPi . The ®lter was rinsed ®ve times with 2 ml 5 % TCA containing 1 % PPi , twice with 2 ml 100 % ethanol and air dried. The remaining radioactivity was counted in a beta scintillation counter using 10 ml scintillation solution. DNase II activity was measured in 200 ml 10 mM Tris±HCl buffer containing 10 mM EDTA pH 5.75. The reaction was allowed to proceed and the nondigested DNA was measured as for DNase I. Preparation of Retina Extracts for DNase Activities Measurements Eyes from E6, E11, E18 and PH8 were enucleated and the retina were dissected under a binocular microscope. Three hundred mg (average) of tissues were homogenized in 6 ml 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 7.4. The homogenates obtained were centrifuged for 30 min at 48C at 10 000 rpm in a JA 21 centrifuge and then dialysed against 25 mM Tris, 1 mM EDTA, overnight at 48C. The protein concentration was measured by the BCA reagent method (Pierce). Polyclonal Anti-L-DNase II and Anti-DNase I Polyclonal anti-L-DNase II was prepared and fully characterized, as described in Torriglia et al. (1995). This antibody recognizes L-DNase II in its fully processed or intermediate form (27 and 35 kDa, respectively) as well as its precursor, the leukocyte elastase inhibitor (LEI) (42 kDa), and the complex between LEI and elastase (60 kDa) (Christensen et al., 1995; Torriglia et al., 1998). This antibody completely inhibits 4 U of L-DNase II at a dilution of 1/100. Anti-DNase I, commercially available antibody inhibits completely 1 U of DNase I at a dilution of 1/250. Electrophoresis and Immunoblotting Detection Protein samples for electrophoresis were prepared according to Laemmli (1970). Gel electrophoresis was conducted at a constant current of 20 mA using either 12 or 15 % polyacrylamide slab gels. Prestained markers were used for gel calibration. Transfer of
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proteins to Immobilon P membranes was carried out electrophoretically (Towbin, Staehelin and Gordon, 1979). A constant voltage of 40 V was applied for 16 hr. Immediately after blotting, the membranes were soaked for 2 hr at 378C in phosphate buffered saline (PBS, 19 mM Na2HPO4 , 1 mM KH2PO4 , 140 mM NaCl, 15 mM KCl, pH 7.5) containing 5 % fat free dried milk. Afterwards the blot was incubated for 1 hr at room temperature with anti-DNase I antibody (80 mg ml ÿ1) or anti-L-DNase II antibody (1/250) diluted in PBS containing 0.5 % fat free dried milk. Controls included the substitution of equivalent dilution of rabbit preimmune serum for the antisera. Binding of the antibodies was visualized after two washings (10 min each) with PBS containing 0.5 % fat free dried milk, followed by 1 hr incubation at room temperature with peroxidase-conjugated goat anti-rabbit IgG (1 mg ml ÿ1) in PBS containing 0.5 % fatty free dried milk. The sheets were then rinsed successively with 0.5 % fatty free dried milk in PBS (2 5 min), PBS containing 0.1 % Tween 20 (2 5 min) and ®nally with PBS (2 5 min). Peroxidase activity was detected using the ECL method as described (Torriglia et al., 1995). Nuclei Puri®cation One hundred and sixty mg (average) retina at different developmental stages were homogenized in 1.5 ml buffer A (A: 60 mM KCl, 15 mM NaCl, 0.15 mM spermine, 0.05 mM spermidine, 15 mM Tris±HCl, pH 7.4, 0.34M sucrose, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 12.5 mM 2-mercaptoethanol) using a Dounce A manual homogenizer, eight strokes at 08C. The homogenate was then centrifuged in a Heraeus centrifuge at 1500 rpm for 10 min at 48C. The supernatant was discarded and the pellet was resuspended in the same volume of A buffer, homogenized again and centrifuged as before. The resulting pellet was resuspended in the same volume of A and rehomogenized using a Dounce B, four strokes at 08C. The homogenate was centrifuged in a Heraeus centrifuge at 3000 rpm for 10 min at 48C. The pellet was solubilized in 5 ml B buffer, containing 0.34 M sucrose (B: 60 mM KCl, 3 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM PMSF, 12 mM 2-mercaptoethanol, 15 mM Tris±HCl pH 7.4) and centrifuged as before. The resulting pellet was washed once by centrifugation in the same buffer and then resuspended in 0.7 ml B containing 0.34 M sucrose and combined with 1.3 ml B containing 1.38 M sucrose. The resulting mixture was overlaid on the top of a discontinuous sucrose gradient formed by a cushion of 1 ml 1.83 M sucrose in B, placed on the bottom of a SW50 rotor tube (Beckman) and a second layer of 2 ml 1.38 M sucrose in B. The discontinuous sucrose gradient was then centrifuged at 40 000 g for 45 min at 48C. The pellet from this gradient was then solubilized with 0.8 ml B containing 0.34 M sucrose.
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The nuclei were counted with a Malassez chamber. Aliquots of 2 107 nuclei were separated and stored at ÿ208C until use. Immunohistochemistry Eyes from chick embryos were dissected at different stages of development (E6, E11 and E18) and of 1 week post-hatch (PH8). The posterior part of the eye was ®xed with 4 % paraformaldehyde in phosphate buffer saline (PBS) for 2 hr, mounted in Tissue Tek OCT and stored at ÿ808C until use. Serial sections (10 mm thick) were prepared using a Bright OTF/AS cryostat (DIS, Blanc-Mesnil, France), collected on gelatincoated slides and stored at ÿ208C. After removal of OCT from sections, the sections were permeabilized with 0.3 % Triton X-100 in PBS for 30 min. After washing with PBS, sections were saturated with PBS containing 5 % skim milk for 30 min, and then incubated at 48C overnight with either polyclonal DNase I or DNase II antibodies diluted (1/100) in PBS containing 1 % skim milk. After incubation with the speci®c primary antibodies, the sections were washed ®ve times with PBS containing 1 % skim milk and then incubated for 1 hr at room temperature with rhodamine isothiocyanate anti-rabbit IgG (diluted 1 : 100 in PBS per 1 % skim milk). Sections were then extensively washed in PBS and, during the last wash, treated for 5 min with the ¯uorescent nuclear stain DAPI. Sections were then mounted in Fluoprep, viewed under a Leitz Aristoplan microscope equipped with an epi-illuminator HBO and ®lters for rhodamine and DAPI ¯uorescence. 3. Results DNase Activity During Retina Development In order to identify nucleases implicated in retinal apoptosis we measured the activity of different enzymes at different stages of development of the chick retina. Three embryonic stages (E6, E11 and E18) and 1 week old chicken (PH8) were studied. Neural retinas were dissected and the DNase activity measured in the extracts. Representative results of these experiments are shown in Fig. 1(A). In whole retinas, the Ca2 -Mg2 -dependent activity (black bars) was dominated as compared to acidic DNases activities [Fig. 1(A), striped bars]. Ca2 -Mg2 activities seemed to increase at E11, which represents the period of maximal apoptosis in the retina (Ilschner and Warning, 1992). To see if the measured activities were nuclear the same experiments were performed on puri®ed nuclei. The activity accumulated in nuclei was almost not detectable during development, except at E11 and E18 [Fig. 1(B)]. At E11 a huge increase was observed (note that the scale is different for whole retinas and for nuclei extracts). This increase implicated
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DNase I in Retina Development
F IG . 1. DNase activity in chicken retina, (A) total retina activity, (B) puri®ed nuclei activity and (C) percent of nuclear activity. One mg 3H-labeled DNA was incubated in the presence of whole retina or puri®ed nuclei extracts for 30 min at 378C. Different buffers were used to measure different DNases: 10 mM Tris±HCl, pH 7.4 containing 10 mM CaCl2, 10 mM MgCl2, Ca2 -Mg2-dependent DNases (j): 10 mM Tris, 10 mM EDTA, pH 5.5 (acid DNases) (F). Undigested DNA was TCA-precipitated and measured in a scintillation counter. DNase speci®c activity was expressed as ng DNA digested min ÿ1 mg ÿ1 protein in (A) and (B). In (C) the total and nuclear activities were referred to the amount of tissue that generates these activities. The nuclear activity is represented.
Ca2 -Mg2 -dependent DNases, as well as acid DNases. Moreover, if the DNase activity generated by nuclear or total extracts is reported to the amount of tissue that produced this activity, we can calculate the percent of activity located in the nuclear fraction at each stage. Fig. 1(C) shows that only at E11 and E18 a signi®cant amount of DNase activity is nuclear. At E11 almost 100 % of the activity measured was nuclear. This result suggest that both Ca2 -Mg2 -dependent and acid DNases activity were related to the apoptotic process.
As Ca2 -Mg2 -dependent DNases represent a family of enzymes, we investigated using commercially available antibodies, whether DNase I, the best characterized Ca2 -Mg2 -dependent DNase (Peitsch et al., 1993), could be responsible for this activity. Its presence was determined by Western blot using whole retinas or puri®ed nuclei (Fig. 2). This antibody was shown to be able to recognize this enzyme in Western blot and Elisa. It immunoprecipitated DNase I and it inhibited its endonuclease activity (not shown) (Torriglia et al., 1995). DNase I was not detectable by Western blots of total retinal extracts [Fig. 2(A)], but two immunoreactive bands were detected in puri®ed nuclei, and altered with differentiation [Fig. 2(B)]. It is worthwhile to note that a band of about 32 kDa, the molecular weight of active DNase I, appeared at E11. All these bands disappeared when the antibody was exhausted with DNase I (not shown). The labelling obtained at higher molecular weight (45 and 60 kDa approximately) observed in Fig. 2(B) may represent DNase I bounded to other proteins, like actin, a protein that binds and inhibits DNase I (Perez, Arner and Hakansson, 1997; Kim et al., 2000) or proteins cross-reacting with DNase 1. It is possible that the absence of bands in total retina extracts was due to a lack of sensitivity. One hundred mg of proteins per line were loaded when using total extracts and 35 mg when analysing nuclear extracts. As nuclear extracts represented roughly 5 % of total extracts, one may assume that in the Western blot seen in Fig. 2(A) only 5 mg of nuclear proteins have been charged, that is, seven-fold less than on the blot shown on Fig. 2(B). The nuclear location of DNase I was further investigated by immunohistochemistry (Fig. 3). In retinal sections, DNase I immunoreactivity was localized all over the neuro-epithelium at E6. The nuclear staining with DAPI seemed to be complementary to the antibody labelling, suggesting a cytoplasmic location of DNase I. Later in the development, the labelling became nuclear. At E11 some nuclei of the outer nuclear layer were labelled, as well as most ganglion cell nuclei and some intermediate neurons. This distribution pattern of the labelling may be correlated with apoptosis that concerns, at E11, the ganglion and intermediate cells layers. At E18 apoptosis concerns mostly the intermediate cell layer but at this stage, the ganglion cell layer is also labelled. Moreover, almost no cells die at PH8 but we still have some labelling. According to Western blot, the labelling found at stages other than E11 might correspond to the higher molecular weight bands. If we correlate this labeling with the decrease of activity found at these late stages (Fig. 1) we may assume that it represents inactive DNase I. In conclusion, studies performed using antibodies directed against DNase I indicate that the increase of
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F IG . 2. Western blot analysis of DNase I in total retina extracts (A) and in extracts from puri®ed nuclei (B). Proteins from total retina (100 mg per line) or puri®ed retina nuclei (3 106 nuclei, about 35 mg of protein per line) were separated in 12 % acrylamide slab gel, then transferred to Immobilon P and analysed using an anti-DNase I antibody. E6, E11 and E18 represents extracts from these embryonic days. PH8 represents extracts performed at 8 days post-hatched. The arrow head indicates the 32 kDa band (the size expected for DNase I) seen at E11 in puri®ed nuclei.
Ca -Mg -dependent activity seen in Fig. 1 might correspond to DNase I. L-DNase II in Retina Development The same studies were performed using an antibody directed against L-DNase II, an acid DNase. This antibody was prepared and fully characterized in our laboratory (Torriglia et al., 1995). It bears reminding that L-DNase II derives from a precursor, LEI (Torriglia et al., 1998), so that the antibody reacts with several forms of this molecule. Brie¯y, several bands of different apparent molecular weights might be expected: L-DNase II in its fully processed or intermediate form (27 and 35 kDa, respectively) as well as its precursor, LEI (42 kDa), and the complex between LEI and elastase (60 kDa) (Teschauer, Mentele and Sommerhoff, 1993; Christensen et al., 1995). Western blots analysing extracts from total retina or puri®ed nuclei are shown in Fig. 4(A) and (B), respectively. In total retina extracts, no immunoreactive band was seen at E6. In contrast, at E11 a band of 35 kDa was present (arrow). The intensity of this band increased slightly at E18 then decreased at PH8. At E18 and PH8, two other bands of 42 and 60 kDa were also present. Also, a very faint band of 92 kDa was present from E11 to PH8. At the nuclear level (B), a band of approximately 92 kDa was seen at all the embryonic stages but not in post-hatched chickens. At E11, two bands of 35 (arrow) and 42 kDa were observed. The 35 kDa band was also seen at E18, while the 42 kDa band had disappeared completely. Control blots (not shown) using antiL-DNase II preimmune show no labelling, indicating that all these bands were speci®c. So, all the bands found were expected except for the 92 kDa band that is of an unknown nature but that we have already seen in apoptotic cells (unpublished data). It is interesting to note that the fully active form of
L-DNase II (27 kDa) is not seen in retina. This might be due to a short half-life of this form since its activity can be measured. Western blot results indicate the presence of active forms of L-DNase II at E11 and E18 in equivalent amounts, however activity measurements show higher activity at E11. This suggests that this activity might be regulated by other factors. In fact, inhibitors of DNase II activity have already been described (Lesca and Paoletti, 1969; Lesca, 1976). The nuclear localization of L-DNase II was veri®ed by indirect immunohistochemistry. The results obtained are depicted in Fig. 5. The immunolabelling was cytoplasmic in the neuro-epithelium at E6. Then, at E11, we observed an accumulation of immunoreactivity in the inner nuclear layer, as well as in photoreceptor nuclei and in ganglion cell nuclei. At E18, nuclei of ganglion cells were labeled, as well as photoreceptor nuclei and the inner area of the inner nuclear layer. A labeling of the plexiform layers was seen mostly in the inner plexiform layer. The same kind of immunolabeling was detected at PH8, except that the reactivity of the inner plexiform layer was fainter. The nuclei of the ganglion cell layer remained labeled, as well as photoreceptors nuclei. If the distribution pattern of L-DNase II is compared to the one of DNase I, little difference is seen. Both enzymes follow roughly the same distribution over development. Are DNase I and L-DNase II Degrading DNA at an Equivalent Extent? The results described above indicated the presence and the activation in the developing retina of both Ca2 -Mg2 -dependent DNases, like DNase I and acid DNases like L-DNase II. To decide which of these enzymes were involved in nuclear degradation during retina apoptosis, we puri®ed retinal nuclei at the different embryonic and post-hatched stages studied,
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F IG . 3. Immunolocalization of DNase I in chick retina. Transverse sections of retinas from E6, E11 and E18 and PH8 were incubated with a polyclonal antibody against DNase I and photographed using a rhodamine ®lter. The same sections were treated with DAPI and observed with an appropriate ®lter to determine the location of the different nuclear layers. The same experiments were performed using the same serum exhausted with DNase I (exhausted anti-DNase I). With the exhausted serum no labelling was observed. NE: neuro-epithelium, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. White bar represents 60 mm.
F IG . 4. Western blot analysis of L-DNase II in total retina extracts (A) and in extracts from puri®ed nuclei (B). Proteins from total retina (100 mg per line) or puri®ed retina nuclei (3 106 nuclei, about 35 mg of protein per line) were separated in 12 % acrylamide slab gel, then transferred to Immobilon P and analysed with an anti-L-DNase II antibody. E6, E11, E18 represents extracts from these embryonic days. PH8 represents extracts from 8 days post-hatching. Arrows indicate the 35 kDa band.
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F IG . 5. Immunolocalization of L-DNase II in chick retina. Transverse sections of retinas from E6, E11 and E18 and PH8 were incubated with polyclonal antibody against L-DNase II and photographed using a rhodamine ®lter. The same slices were treated with DAPI and observed with an appropriate ®lter to see the location of the different nuclear layers. Control experiments were performed using the pre-immune serum. NE: neuro-epithelium, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. White bar represents 60 mm.
and incubated under either Ca2 -Mg2-DNase or L-DNase II activating conditions. This study (Fig. 6) was performed using retina cell nuclei from E11 and E18 because Western blot analysis and activity measurements showed (Figs 1, 2 and 4) that the active forms of DNase I and L-DNase II were present only at these stages in nuclei. In the presence of cations the DNA was degraded to a certain extent. This degradation was not inhibited by the anti-DNase I antibody when used at a 1/10 dilution (shown in Fig. 6) or 1/20 dilution (not shown), even if this antibody fully inhibits 1 U of DNase I at 1/250 dilution and inhibits the cleavage of DNA from retina when 1 U of DNase I is added to puri®ed retina nuclei (not shown). A similar situation was noted when using acid DNase activating conditions at E18, that is to say, the presence of anti-L-DNase II did not change the degradation pattern. A more extensive degradation of DNA was seen at E11 in acidic conditions. This degradation was impaired when L-DNase II antibody was added to the reaction mixture. In this
case, the degradation of DNA seems less important than in the control DNA. This indicates that L-DNase II is bound to DNA. We have already seen this gel retard effect when antibodies are added after the binding of L-DNase II to DNA `in vitro' (Torriglia et al., 1998). This gel retard effect is less strong at E18, indicating that, at this stage, most DNA is L-DNase II-free. The results shown in Fig. 6 indicated that from the two families of DNases measured, acid DNases were more active on retinal DNA than Ca -Mg -dependent DNases. Moreover, if anti-L-DNase II seemed ef®ciently protected DNA, anti-DNase I had no effect. 4. Discussion Cell proliferation has been extensively studied in embryonic development. Over the past years it has become evident that cell death is as important as proliferation in tissue histogenesis. In the central nervous system, apoptosis is implicated in neural
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F IG . 6. Effect of the anti-DNase I and anti-L-DNase II antibodies on DNA cleavage. 106 nuclei from retina at embryonic stages (E11 and E18), were incubated in a neutral cationic medium (lanes DNase I), alone (lane ÿ) or in the presence of anti-DNase I (lane ). In lanes `L-DNase II', an acidic medium was used. The experiment was performed in the absence (ÿ) or in the presence () of anti-L-DNase II. Control lanes represent the state of DNA after puri®cation of the nuclei. Arrow heads show the position of the sample wells.
death, a mechanism used to build the neuronal network (Sherrard and Bower, 1998). The survival of developing neurons depends on interactions with other neurons and glial cells (Linden, 1994; Sherrard and Bower, 1998). Recent work has shown that, in addition to neurotrophic peptides, neurotransmitters and other metabolites affect the survival of retinal neurons: changes in the levels of nitric oxide, catecholamine metabolism, or glutamate receptor activation may trigger apoptosis in retinal cells (Goureau et al., 1993; Meyer-Franke et al., 1995; Perez et al., 1997). Studies in Caenorhabitis elegans were very important in providing a molecular framework for the cell death pathway (Bergmann, Agapite and Steller, 1998) but recent studies have shown that the molecular pathways of apoptosis are more complex in high vertebrates and mammals (Wilson, 1998), where many pathways may be activated. A common feature of the activation of the different apoptotic pathways seems to be the activation of degradative enzymes like proteases and endonucleases that degrade vital molecules. Thus, the activation of different proteases or endonucleases may re¯ect the activation of different apoptotic pathways. Several DNases have been implicated in apoptosis. A number of cationic DNases acting at neutral pH have been characterized, such as in thymocytes, NUC 18/ cyclophilin A (Montague et al., 1994), DNase I (Peitsch et al., 1993; Stephen et al., 1996), DNase g (Shiokawa et al., 1994), and a 97 kDa DNase (Pandey, Walker and Sikorska, 1997). In human myeloid cell
lines, Mg2 -dependent, Ca2 -independent DNases have been identi®ed (Kawabata et al., 1993, 1997). Among these DNases, the most well characterized is CAD, a Caspase Activated DNase (Enari et al., 1998; Halenbeck et al., 1998). Barry and Eastman (1992) have described in CHO cells another class of DNase capable of playing a role in the apoptotic DNA digestion, that is acid and non-cationic DNase II. This enzyme is activated by intracellular acidi®cation which occurs during apoptosis (PeÂrez-Sala, ColladoEscobar and Mollinedo, 1995; Wolf, Morana and Eastman, 1997). We have also observed a DNase II implicated in DNA degradation during chick lens cell differentiation (Torriglia et al., 1995). Further characterization of this DNase II has shown that this 27 kDapeptide is derived, by a post-translational modi®cation, from a precursor of 42 kDa (Torriglia et al., 1998). This precursor, in its native form, is identical to the leukocyte elastase inhibitor (LEI). The anti-protease activity of LEI is lost during its conversion to active DNase II, which is a protein of 35 kDa, before its interaction with DNA, after which it is converted to a 27 kDa protein. As this DNase II is different from other DNases II recently described (Baker et al., 1998; Krieser and Eastman, 1998; Shiokawa and Tanuma, 1998; Wang et al., 1998; Yasuda et al., 1998), and as it is derived from LEI, we name it L-DNase II. In this paper, we have investigated the DNases which may be activated during apoptosis occurring during embryonic development of the retina. Two classes of DNases were considered: Ca2 -Mg2-dependent and acid
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DNases. We did not include CAD since this DNase does not seem to be expressed in the central nervous system (Mukae et al., 1998). The experiments discussed above show the presence of several DNases activated in the retina during embryonic development which could be responsible for DNA degradation in neurons dying by apoptosis. Ca2 -Mg2-dependent and acid nucleases are activated when apoptosis peaks. Results obtained by immunological methods indicate that both types of enzymes are located in the nucleus in their active forms. To elucidate if there is a difference in the involvement of these DNases, we incubated puri®ed nuclei under Ca2 -Mg2 and acid DNase-activating conditions in the presence or in the absence of anti-DNase I or antiL-DNase II antibodies which determined the resident DNases involved in DNA degradation. According to activity measurements, the highest degradation was obtained in nuclei from E11 retinas but degradation was maximal under acid DNases activating conditions. Moreover, DNA was protected when the experiment was done in the presence of antiL-DNase II. These results indicate that even if other nucleases bearing DNase II activity may exist or if DNA could be degraded by the acidic conditions used, L-DNase II seems responsible for most of DNA degradation in these nuclei. In addition, in spite of the presence of DNase I in retina nuclei at E11, its participation to DNA degradation is not very important since no protection is obtained with anti-DNase I, an antibody that inhibits DNase I activity (Torriglia et al., 1995). This does not rule out completely the participation of DNase I but strongly suggests that other DNases Ca2 -Mg2 -dependent different than DNase I may be involved. From the different Ca2 Mg2-dependent DNases implicated in apoptosis some of them, like DNase g (Shiokawa, Iwamatsu and Tanuma, 1997), for instance, have been fully characterized and their involvement in retina apoptosis is under current investigation. The results obtained for the developing retina are similar to those obtained in the lens (Torriglia et al., 1995) and in other cell models, like the HeLa cells, when apoptosis has been induced by long term culture (Torriglia et al., 1999). In HeLa cells the activation of the L-DNase pathway does not impair the activation of other nucleases at the same time, just like in the developing retina. We may then presume that the L-DNase II apoptotic pathway is activated when cells are not receiving the extracellular signals to survive (Jacobson, Weil and Raff, 1997). However, this is not the only pathway activated. Other apoptotic pathways seem to be important to retina development, such as, the caspasedependent pathways. Actually, the inactivation of caspase 3, via gene inactivation of Apaf-1 generates defects in retina development (Cecconi et al., 1998). However 5 % of these mutants survive and the central nervous system develops normally (Honarpour et al.,
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2000). It is thus possible that different apoptotic pathways are activated in individual cells according to the speci®c signal that triggers apoptosis in the cell. Therefore, several apoptotic pathways may run concurrently to regulate cell number. Acknowledgements We acknowledge Dr Jean Claude Jeanny and Laurent Jonet for the sections, Dr Lisa Oliver for correcting the English manuscript, and Herve Coet for the photographs. This work was supported by INSERM, APEX contract 4X012D and by Retina France.
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Involvement of L-DNase II in Nuclear Degeneration during Chick Retina Development AL IC IA TORR IGL IA* , E L I S A B E T H C H A U D U N , F R A N CË O I S E C H A N Y - FO U R N I E R , Y V E S CO U R TO I S A N D M A R IE - F R A N C E CO U N I S Unite 450 INSERM, Af®lieÂe CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France (Received Milwaukee 21 June 2000, accepted in revised form 8 December 2000 and published electronically 8 February 2001) During the development of the neural retina, 50 % of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the ®nal event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2 -Mg2 -dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using puri®ed nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase # 2001 Academic Press is responsible for most of the DNA degradation observed in these cells. Key words: DNases; retina development; apoptosis; L-DNase II; DNase I.
1. Introduction The retina is derived from the central nervous system, from an invagination of the proencephalin, called the optical vesicle. In the early stages of embryonic development, the retina is composed of undifferentiated neuro-epithelial cells. As the embryonic development proceeds the neuro-epithelium differentiates into three layers (Cepko, 1984). The outer layer contains the sensorial cells, the photoreceptors; the inner nuclear layer is formed by the ganglion cells. Between these two layers, the nuclei of the intermediate neurons are found. These nuclear layers are separated by the so-called plexiform layers which contain the synaptic regions and the axonal extensions. During the onset of this differentiation, the neurons which fail to establish the right synaptic connections are eliminated. In chick embryo, the retinal organogenesis starts at E8 and is almost complete at E18. During this period almost 50 % of the cells die. This cellular death occurs by the mechanism of apoptosis (Young, 1984). Waves of apoptosis sweep through the ganglion cell layer, inner nuclear layer and ®nally the photoreceptor layer as each region undergoes differentiation (Ilschner and Warning, 1992; Prindull, 1995). Apoptosis was described by Wyllie (1980) as a unique form of cell death that could be distinguished from necrosis induced by toxins and anoxic injury. The morphological hallmarks include cytoplasmic and nuclear condensation and fragmentation of cells into membrane-bound compartments (apoptotic * Author for correspondence. E-mail: [email protected]
0014-4835/01/04044311 $35.00/0
bodies) (Leach, 1998). These apoptotic cells are phagocytozed by adjacent cells and occasionally by resident macrophages. According to the current concepts, apoptosis is triggered by diverse interacting signals including expression of oncogenes (e.g. bcl-2, c-myc) (Yang and Korsmeyer, 1996; Prendergast, 1999) and tumor suppressor genes (Choisy-Rossi and Yonish-Rouach, 1998; Kasten and Giordano, 1998). As the molecular mechanism of apoptosis begin to be elucidated, the morphological criteria have been replaced by biochemical indicators such as DNA fragmentation, visualized in agarose gel electrophoresis as the `oligonucleosomal ladder' (Collins et al., 1992; Walker and Sikorska, 1995). Nucleosomal laddering is now viewed as a late and inconstant sign of apoptosis (Collins et al., 1992). Earlier stages are evidenced by high molecular DNA fragments (Brown, Sun and Cohen, 1993; Oberhammer et al., 1993). This cleavage of the genomic DNA is performed by endonucleases. Many studies have suggested that this is a Ca2 -dependent process (Gaido and Cidlowski, 1991; Nicotera and Rossi, 1994), but Kluck et al. (1994) have shown several instances where apoptosis was not associated with elevated intracellular Ca2 levels. Barry and Eastman proposed that under certain conditions DNase II, a Ca2 -independent DNase, may be activated by intracellular acidi®cation (Barry and Eastman, 1992; Eastman, 1994). Recent studies performed in our laboratory, analysing terminal differentiation in lens cells, seem to con®rm this conclusion (Torriglia et al., 1995; Counis et al., 1998). We have recently characterized the DNase responsible for this degradation. # 2001 Academic Press
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This enzyme has a DNase II-like activity, i.e. it is Ca2 Mg2 -independent and presents an optimal activity at acidic pH. This DNase is derived by post-translational modi®cation of the leukocyte elastase inhibitor (LEI) and it is called L-DNase II (Torriglia et al., 1998). In this study we tried to identify the endonuclease activated during apoptosis in the developing retina. This is an important piece of knowledge to investigate the apoptotic pathways activated to eliminate useless cells. We have identi®ed Ca2 -Mg2 -dependent and acidic endonucleases, in the developing chick retina, by measuring the activity of both enzymes in the total and nuclear extracts and by using antibodies against DNase I and L-DNase II. We show that both DNase I and L-DNase II, as well as other Ca2 -Mg2 dependent DNases are activated. DNase I does not seem to play a major role, but the implication of L-DNase II and the activation of its speci®c pathway is discussed.
2. Materials and Methods Materials Fertilized eggs of white Leghorn chicken were obtained from Hass (Kalthenhouse, France). Calf thymus DNA was from Pharmacia. Puri®ed DNase I was from Sigma and L-DNase II was from Worthington. Deoxy [8-3H] adenosine 50 triphosphate, ammonium salt (code TRK 34, 777 GBq mmol ÿ1) and enhanced chemiluminescence kit (ECL) were from Amersham International. Immobilon P was from Millipore. Escherichia coli DNA polymerase I (endonuclease free), dCTP, dGTP, dTTP were from Boehringher Mannheim. Polyclonal antibody against DNase I was from Rockland and biotinylated goat anti-rabbit IgG were from Cooper Biomedica. The scintillation liquid was Luma Safe from Packard. Prestained molecular weight markers for electrophoresis were from BioRad. The mounting material, Tissue Tek OCT, was obtained from Miles. The polyclonal antibody directed against DNase I was from Rockland. DNA Labeling Calf thymus DNA (0.5 mg) was 3H-labeled in 10 ml 50 mM Tris±HCl, pH 7.4 containing 5 mM MgCl2 , 50 mg ml ÿ1 bovine serum albumin, 10 mM 2mercaptoethanol, 50 mM dCTP, dGTP and dTTP, 2.5 nM 3HdATP and 100 IU DNA polymerase I. The reaction was allowed to proceed for 30 min at 158C then stopped with the addition of NaCl to a concentration of 150 mM. The labeled DNA was then precipitated with two volumes of 100 % ethanol for 2 hr at 08C. The precipitated DNA was washed four times with 70 % ethanol and centrifuged each time at 2500 rpm for 10 min. After air drying of the pellet, the labeled DNA was solubilized at 48C in 1 ml
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H2O. The concentration of solubilized DNA was evaluated by spectrophotometric reading at 260 nm. Measurement of DNase Activities DNase I activity was measured in 200 ml 10 mM Tris±HCl pH 7.4 containing 10 mM CaCl2 and 10 mM MgCl2 . The reaction, started with 1 mg 3H-DNA, was allowed to proceed for 30 min at 378C, then stopped by incubation for 1 hr at 08C with 200 ml of 10 % trichloroacetic acid (TCA) containing 1 % sodium pyrophosphate (PPi). The nondigested DNA was then trapped on a GF/C ®lter (Millipore) previously soaked in 1 % PPi . The ®lter was rinsed ®ve times with 2 ml 5 % TCA containing 1 % PPi , twice with 2 ml 100 % ethanol and air dried. The remaining radioactivity was counted in a beta scintillation counter using 10 ml scintillation solution. DNase II activity was measured in 200 ml 10 mM Tris±HCl buffer containing 10 mM EDTA pH 5.75. The reaction was allowed to proceed and the nondigested DNA was measured as for DNase I. Preparation of Retina Extracts for DNase Activities Measurements Eyes from E6, E11, E18 and PH8 were enucleated and the retina were dissected under a binocular microscope. Three hundred mg (average) of tissues were homogenized in 6 ml 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 7.4. The homogenates obtained were centrifuged for 30 min at 48C at 10 000 rpm in a JA 21 centrifuge and then dialysed against 25 mM Tris, 1 mM EDTA, overnight at 48C. The protein concentration was measured by the BCA reagent method (Pierce). Polyclonal Anti-L-DNase II and Anti-DNase I Polyclonal anti-L-DNase II was prepared and fully characterized, as described in Torriglia et al. (1995). This antibody recognizes L-DNase II in its fully processed or intermediate form (27 and 35 kDa, respectively) as well as its precursor, the leukocyte elastase inhibitor (LEI) (42 kDa), and the complex between LEI and elastase (60 kDa) (Christensen et al., 1995; Torriglia et al., 1998). This antibody completely inhibits 4 U of L-DNase II at a dilution of 1/100. Anti-DNase I, commercially available antibody inhibits completely 1 U of DNase I at a dilution of 1/250. Electrophoresis and Immunoblotting Detection Protein samples for electrophoresis were prepared according to Laemmli (1970). Gel electrophoresis was conducted at a constant current of 20 mA using either 12 or 15 % polyacrylamide slab gels. Prestained markers were used for gel calibration. Transfer of
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proteins to Immobilon P membranes was carried out electrophoretically (Towbin, Staehelin and Gordon, 1979). A constant voltage of 40 V was applied for 16 hr. Immediately after blotting, the membranes were soaked for 2 hr at 378C in phosphate buffered saline (PBS, 19 mM Na2HPO4 , 1 mM KH2PO4 , 140 mM NaCl, 15 mM KCl, pH 7.5) containing 5 % fat free dried milk. Afterwards the blot was incubated for 1 hr at room temperature with anti-DNase I antibody (80 mg ml ÿ1) or anti-L-DNase II antibody (1/250) diluted in PBS containing 0.5 % fat free dried milk. Controls included the substitution of equivalent dilution of rabbit preimmune serum for the antisera. Binding of the antibodies was visualized after two washings (10 min each) with PBS containing 0.5 % fat free dried milk, followed by 1 hr incubation at room temperature with peroxidase-conjugated goat anti-rabbit IgG (1 mg ml ÿ1) in PBS containing 0.5 % fatty free dried milk. The sheets were then rinsed successively with 0.5 % fatty free dried milk in PBS (2 5 min), PBS containing 0.1 % Tween 20 (2 5 min) and ®nally with PBS (2 5 min). Peroxidase activity was detected using the ECL method as described (Torriglia et al., 1995). Nuclei Puri®cation One hundred and sixty mg (average) retina at different developmental stages were homogenized in 1.5 ml buffer A (A: 60 mM KCl, 15 mM NaCl, 0.15 mM spermine, 0.05 mM spermidine, 15 mM Tris±HCl, pH 7.4, 0.34M sucrose, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 12.5 mM 2-mercaptoethanol) using a Dounce A manual homogenizer, eight strokes at 08C. The homogenate was then centrifuged in a Heraeus centrifuge at 1500 rpm for 10 min at 48C. The supernatant was discarded and the pellet was resuspended in the same volume of A buffer, homogenized again and centrifuged as before. The resulting pellet was resuspended in the same volume of A and rehomogenized using a Dounce B, four strokes at 08C. The homogenate was centrifuged in a Heraeus centrifuge at 3000 rpm for 10 min at 48C. The pellet was solubilized in 5 ml B buffer, containing 0.34 M sucrose (B: 60 mM KCl, 3 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM PMSF, 12 mM 2-mercaptoethanol, 15 mM Tris±HCl pH 7.4) and centrifuged as before. The resulting pellet was washed once by centrifugation in the same buffer and then resuspended in 0.7 ml B containing 0.34 M sucrose and combined with 1.3 ml B containing 1.38 M sucrose. The resulting mixture was overlaid on the top of a discontinuous sucrose gradient formed by a cushion of 1 ml 1.83 M sucrose in B, placed on the bottom of a SW50 rotor tube (Beckman) and a second layer of 2 ml 1.38 M sucrose in B. The discontinuous sucrose gradient was then centrifuged at 40 000 g for 45 min at 48C. The pellet from this gradient was then solubilized with 0.8 ml B containing 0.34 M sucrose.
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The nuclei were counted with a Malassez chamber. Aliquots of 2 107 nuclei were separated and stored at ÿ208C until use. Immunohistochemistry Eyes from chick embryos were dissected at different stages of development (E6, E11 and E18) and of 1 week post-hatch (PH8). The posterior part of the eye was ®xed with 4 % paraformaldehyde in phosphate buffer saline (PBS) for 2 hr, mounted in Tissue Tek OCT and stored at ÿ808C until use. Serial sections (10 mm thick) were prepared using a Bright OTF/AS cryostat (DIS, Blanc-Mesnil, France), collected on gelatincoated slides and stored at ÿ208C. After removal of OCT from sections, the sections were permeabilized with 0.3 % Triton X-100 in PBS for 30 min. After washing with PBS, sections were saturated with PBS containing 5 % skim milk for 30 min, and then incubated at 48C overnight with either polyclonal DNase I or DNase II antibodies diluted (1/100) in PBS containing 1 % skim milk. After incubation with the speci®c primary antibodies, the sections were washed ®ve times with PBS containing 1 % skim milk and then incubated for 1 hr at room temperature with rhodamine isothiocyanate anti-rabbit IgG (diluted 1 : 100 in PBS per 1 % skim milk). Sections were then extensively washed in PBS and, during the last wash, treated for 5 min with the ¯uorescent nuclear stain DAPI. Sections were then mounted in Fluoprep, viewed under a Leitz Aristoplan microscope equipped with an epi-illuminator HBO and ®lters for rhodamine and DAPI ¯uorescence. 3. Results DNase Activity During Retina Development In order to identify nucleases implicated in retinal apoptosis we measured the activity of different enzymes at different stages of development of the chick retina. Three embryonic stages (E6, E11 and E18) and 1 week old chicken (PH8) were studied. Neural retinas were dissected and the DNase activity measured in the extracts. Representative results of these experiments are shown in Fig. 1(A). In whole retinas, the Ca2 -Mg2 -dependent activity (black bars) was dominated as compared to acidic DNases activities [Fig. 1(A), striped bars]. Ca2 -Mg2 activities seemed to increase at E11, which represents the period of maximal apoptosis in the retina (Ilschner and Warning, 1992). To see if the measured activities were nuclear the same experiments were performed on puri®ed nuclei. The activity accumulated in nuclei was almost not detectable during development, except at E11 and E18 [Fig. 1(B)]. At E11 a huge increase was observed (note that the scale is different for whole retinas and for nuclei extracts). This increase implicated
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DNase I in Retina Development
F IG . 1. DNase activity in chicken retina, (A) total retina activity, (B) puri®ed nuclei activity and (C) percent of nuclear activity. One mg 3H-labeled DNA was incubated in the presence of whole retina or puri®ed nuclei extracts for 30 min at 378C. Different buffers were used to measure different DNases: 10 mM Tris±HCl, pH 7.4 containing 10 mM CaCl2, 10 mM MgCl2, Ca2 -Mg2-dependent DNases (j): 10 mM Tris, 10 mM EDTA, pH 5.5 (acid DNases) (F). Undigested DNA was TCA-precipitated and measured in a scintillation counter. DNase speci®c activity was expressed as ng DNA digested min ÿ1 mg ÿ1 protein in (A) and (B). In (C) the total and nuclear activities were referred to the amount of tissue that generates these activities. The nuclear activity is represented.
Ca2 -Mg2 -dependent DNases, as well as acid DNases. Moreover, if the DNase activity generated by nuclear or total extracts is reported to the amount of tissue that produced this activity, we can calculate the percent of activity located in the nuclear fraction at each stage. Fig. 1(C) shows that only at E11 and E18 a signi®cant amount of DNase activity is nuclear. At E11 almost 100 % of the activity measured was nuclear. This result suggest that both Ca2 -Mg2 -dependent and acid DNases activity were related to the apoptotic process.
As Ca2 -Mg2 -dependent DNases represent a family of enzymes, we investigated using commercially available antibodies, whether DNase I, the best characterized Ca2 -Mg2 -dependent DNase (Peitsch et al., 1993), could be responsible for this activity. Its presence was determined by Western blot using whole retinas or puri®ed nuclei (Fig. 2). This antibody was shown to be able to recognize this enzyme in Western blot and Elisa. It immunoprecipitated DNase I and it inhibited its endonuclease activity (not shown) (Torriglia et al., 1995). DNase I was not detectable by Western blots of total retinal extracts [Fig. 2(A)], but two immunoreactive bands were detected in puri®ed nuclei, and altered with differentiation [Fig. 2(B)]. It is worthwhile to note that a band of about 32 kDa, the molecular weight of active DNase I, appeared at E11. All these bands disappeared when the antibody was exhausted with DNase I (not shown). The labelling obtained at higher molecular weight (45 and 60 kDa approximately) observed in Fig. 2(B) may represent DNase I bounded to other proteins, like actin, a protein that binds and inhibits DNase I (Perez, Arner and Hakansson, 1997; Kim et al., 2000) or proteins cross-reacting with DNase 1. It is possible that the absence of bands in total retina extracts was due to a lack of sensitivity. One hundred mg of proteins per line were loaded when using total extracts and 35 mg when analysing nuclear extracts. As nuclear extracts represented roughly 5 % of total extracts, one may assume that in the Western blot seen in Fig. 2(A) only 5 mg of nuclear proteins have been charged, that is, seven-fold less than on the blot shown on Fig. 2(B). The nuclear location of DNase I was further investigated by immunohistochemistry (Fig. 3). In retinal sections, DNase I immunoreactivity was localized all over the neuro-epithelium at E6. The nuclear staining with DAPI seemed to be complementary to the antibody labelling, suggesting a cytoplasmic location of DNase I. Later in the development, the labelling became nuclear. At E11 some nuclei of the outer nuclear layer were labelled, as well as most ganglion cell nuclei and some intermediate neurons. This distribution pattern of the labelling may be correlated with apoptosis that concerns, at E11, the ganglion and intermediate cells layers. At E18 apoptosis concerns mostly the intermediate cell layer but at this stage, the ganglion cell layer is also labelled. Moreover, almost no cells die at PH8 but we still have some labelling. According to Western blot, the labelling found at stages other than E11 might correspond to the higher molecular weight bands. If we correlate this labeling with the decrease of activity found at these late stages (Fig. 1) we may assume that it represents inactive DNase I. In conclusion, studies performed using antibodies directed against DNase I indicate that the increase of
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F IG . 2. Western blot analysis of DNase I in total retina extracts (A) and in extracts from puri®ed nuclei (B). Proteins from total retina (100 mg per line) or puri®ed retina nuclei (3 106 nuclei, about 35 mg of protein per line) were separated in 12 % acrylamide slab gel, then transferred to Immobilon P and analysed using an anti-DNase I antibody. E6, E11 and E18 represents extracts from these embryonic days. PH8 represents extracts performed at 8 days post-hatched. The arrow head indicates the 32 kDa band (the size expected for DNase I) seen at E11 in puri®ed nuclei.
Ca -Mg -dependent activity seen in Fig. 1 might correspond to DNase I. L-DNase II in Retina Development The same studies were performed using an antibody directed against L-DNase II, an acid DNase. This antibody was prepared and fully characterized in our laboratory (Torriglia et al., 1995). It bears reminding that L-DNase II derives from a precursor, LEI (Torriglia et al., 1998), so that the antibody reacts with several forms of this molecule. Brie¯y, several bands of different apparent molecular weights might be expected: L-DNase II in its fully processed or intermediate form (27 and 35 kDa, respectively) as well as its precursor, LEI (42 kDa), and the complex between LEI and elastase (60 kDa) (Teschauer, Mentele and Sommerhoff, 1993; Christensen et al., 1995). Western blots analysing extracts from total retina or puri®ed nuclei are shown in Fig. 4(A) and (B), respectively. In total retina extracts, no immunoreactive band was seen at E6. In contrast, at E11 a band of 35 kDa was present (arrow). The intensity of this band increased slightly at E18 then decreased at PH8. At E18 and PH8, two other bands of 42 and 60 kDa were also present. Also, a very faint band of 92 kDa was present from E11 to PH8. At the nuclear level (B), a band of approximately 92 kDa was seen at all the embryonic stages but not in post-hatched chickens. At E11, two bands of 35 (arrow) and 42 kDa were observed. The 35 kDa band was also seen at E18, while the 42 kDa band had disappeared completely. Control blots (not shown) using antiL-DNase II preimmune show no labelling, indicating that all these bands were speci®c. So, all the bands found were expected except for the 92 kDa band that is of an unknown nature but that we have already seen in apoptotic cells (unpublished data). It is interesting to note that the fully active form of
L-DNase II (27 kDa) is not seen in retina. This might be due to a short half-life of this form since its activity can be measured. Western blot results indicate the presence of active forms of L-DNase II at E11 and E18 in equivalent amounts, however activity measurements show higher activity at E11. This suggests that this activity might be regulated by other factors. In fact, inhibitors of DNase II activity have already been described (Lesca and Paoletti, 1969; Lesca, 1976). The nuclear localization of L-DNase II was veri®ed by indirect immunohistochemistry. The results obtained are depicted in Fig. 5. The immunolabelling was cytoplasmic in the neuro-epithelium at E6. Then, at E11, we observed an accumulation of immunoreactivity in the inner nuclear layer, as well as in photoreceptor nuclei and in ganglion cell nuclei. At E18, nuclei of ganglion cells were labeled, as well as photoreceptor nuclei and the inner area of the inner nuclear layer. A labeling of the plexiform layers was seen mostly in the inner plexiform layer. The same kind of immunolabeling was detected at PH8, except that the reactivity of the inner plexiform layer was fainter. The nuclei of the ganglion cell layer remained labeled, as well as photoreceptors nuclei. If the distribution pattern of L-DNase II is compared to the one of DNase I, little difference is seen. Both enzymes follow roughly the same distribution over development. Are DNase I and L-DNase II Degrading DNA at an Equivalent Extent? The results described above indicated the presence and the activation in the developing retina of both Ca2 -Mg2 -dependent DNases, like DNase I and acid DNases like L-DNase II. To decide which of these enzymes were involved in nuclear degradation during retina apoptosis, we puri®ed retinal nuclei at the different embryonic and post-hatched stages studied,
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F IG . 3. Immunolocalization of DNase I in chick retina. Transverse sections of retinas from E6, E11 and E18 and PH8 were incubated with a polyclonal antibody against DNase I and photographed using a rhodamine ®lter. The same sections were treated with DAPI and observed with an appropriate ®lter to determine the location of the different nuclear layers. The same experiments were performed using the same serum exhausted with DNase I (exhausted anti-DNase I). With the exhausted serum no labelling was observed. NE: neuro-epithelium, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. White bar represents 60 mm.
F IG . 4. Western blot analysis of L-DNase II in total retina extracts (A) and in extracts from puri®ed nuclei (B). Proteins from total retina (100 mg per line) or puri®ed retina nuclei (3 106 nuclei, about 35 mg of protein per line) were separated in 12 % acrylamide slab gel, then transferred to Immobilon P and analysed with an anti-L-DNase II antibody. E6, E11, E18 represents extracts from these embryonic days. PH8 represents extracts from 8 days post-hatching. Arrows indicate the 35 kDa band.
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F IG . 5. Immunolocalization of L-DNase II in chick retina. Transverse sections of retinas from E6, E11 and E18 and PH8 were incubated with polyclonal antibody against L-DNase II and photographed using a rhodamine ®lter. The same slices were treated with DAPI and observed with an appropriate ®lter to see the location of the different nuclear layers. Control experiments were performed using the pre-immune serum. NE: neuro-epithelium, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. White bar represents 60 mm.
and incubated under either Ca2 -Mg2-DNase or L-DNase II activating conditions. This study (Fig. 6) was performed using retina cell nuclei from E11 and E18 because Western blot analysis and activity measurements showed (Figs 1, 2 and 4) that the active forms of DNase I and L-DNase II were present only at these stages in nuclei. In the presence of cations the DNA was degraded to a certain extent. This degradation was not inhibited by the anti-DNase I antibody when used at a 1/10 dilution (shown in Fig. 6) or 1/20 dilution (not shown), even if this antibody fully inhibits 1 U of DNase I at 1/250 dilution and inhibits the cleavage of DNA from retina when 1 U of DNase I is added to puri®ed retina nuclei (not shown). A similar situation was noted when using acid DNase activating conditions at E18, that is to say, the presence of anti-L-DNase II did not change the degradation pattern. A more extensive degradation of DNA was seen at E11 in acidic conditions. This degradation was impaired when L-DNase II antibody was added to the reaction mixture. In this
case, the degradation of DNA seems less important than in the control DNA. This indicates that L-DNase II is bound to DNA. We have already seen this gel retard effect when antibodies are added after the binding of L-DNase II to DNA `in vitro' (Torriglia et al., 1998). This gel retard effect is less strong at E18, indicating that, at this stage, most DNA is L-DNase II-free. The results shown in Fig. 6 indicated that from the two families of DNases measured, acid DNases were more active on retinal DNA than Ca -Mg -dependent DNases. Moreover, if anti-L-DNase II seemed ef®ciently protected DNA, anti-DNase I had no effect. 4. Discussion Cell proliferation has been extensively studied in embryonic development. Over the past years it has become evident that cell death is as important as proliferation in tissue histogenesis. In the central nervous system, apoptosis is implicated in neural
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F IG . 6. Effect of the anti-DNase I and anti-L-DNase II antibodies on DNA cleavage. 106 nuclei from retina at embryonic stages (E11 and E18), were incubated in a neutral cationic medium (lanes DNase I), alone (lane ÿ) or in the presence of anti-DNase I (lane ). In lanes `L-DNase II', an acidic medium was used. The experiment was performed in the absence (ÿ) or in the presence () of anti-L-DNase II. Control lanes represent the state of DNA after puri®cation of the nuclei. Arrow heads show the position of the sample wells.
death, a mechanism used to build the neuronal network (Sherrard and Bower, 1998). The survival of developing neurons depends on interactions with other neurons and glial cells (Linden, 1994; Sherrard and Bower, 1998). Recent work has shown that, in addition to neurotrophic peptides, neurotransmitters and other metabolites affect the survival of retinal neurons: changes in the levels of nitric oxide, catecholamine metabolism, or glutamate receptor activation may trigger apoptosis in retinal cells (Goureau et al., 1993; Meyer-Franke et al., 1995; Perez et al., 1997). Studies in Caenorhabitis elegans were very important in providing a molecular framework for the cell death pathway (Bergmann, Agapite and Steller, 1998) but recent studies have shown that the molecular pathways of apoptosis are more complex in high vertebrates and mammals (Wilson, 1998), where many pathways may be activated. A common feature of the activation of the different apoptotic pathways seems to be the activation of degradative enzymes like proteases and endonucleases that degrade vital molecules. Thus, the activation of different proteases or endonucleases may re¯ect the activation of different apoptotic pathways. Several DNases have been implicated in apoptosis. A number of cationic DNases acting at neutral pH have been characterized, such as in thymocytes, NUC 18/ cyclophilin A (Montague et al., 1994), DNase I (Peitsch et al., 1993; Stephen et al., 1996), DNase g (Shiokawa et al., 1994), and a 97 kDa DNase (Pandey, Walker and Sikorska, 1997). In human myeloid cell
lines, Mg2 -dependent, Ca2 -independent DNases have been identi®ed (Kawabata et al., 1993, 1997). Among these DNases, the most well characterized is CAD, a Caspase Activated DNase (Enari et al., 1998; Halenbeck et al., 1998). Barry and Eastman (1992) have described in CHO cells another class of DNase capable of playing a role in the apoptotic DNA digestion, that is acid and non-cationic DNase II. This enzyme is activated by intracellular acidi®cation which occurs during apoptosis (PeÂrez-Sala, ColladoEscobar and Mollinedo, 1995; Wolf, Morana and Eastman, 1997). We have also observed a DNase II implicated in DNA degradation during chick lens cell differentiation (Torriglia et al., 1995). Further characterization of this DNase II has shown that this 27 kDapeptide is derived, by a post-translational modi®cation, from a precursor of 42 kDa (Torriglia et al., 1998). This precursor, in its native form, is identical to the leukocyte elastase inhibitor (LEI). The anti-protease activity of LEI is lost during its conversion to active DNase II, which is a protein of 35 kDa, before its interaction with DNA, after which it is converted to a 27 kDa protein. As this DNase II is different from other DNases II recently described (Baker et al., 1998; Krieser and Eastman, 1998; Shiokawa and Tanuma, 1998; Wang et al., 1998; Yasuda et al., 1998), and as it is derived from LEI, we name it L-DNase II. In this paper, we have investigated the DNases which may be activated during apoptosis occurring during embryonic development of the retina. Two classes of DNases were considered: Ca2 -Mg2-dependent and acid
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DNases. We did not include CAD since this DNase does not seem to be expressed in the central nervous system (Mukae et al., 1998). The experiments discussed above show the presence of several DNases activated in the retina during embryonic development which could be responsible for DNA degradation in neurons dying by apoptosis. Ca2 -Mg2-dependent and acid nucleases are activated when apoptosis peaks. Results obtained by immunological methods indicate that both types of enzymes are located in the nucleus in their active forms. To elucidate if there is a difference in the involvement of these DNases, we incubated puri®ed nuclei under Ca2 -Mg2 and acid DNase-activating conditions in the presence or in the absence of anti-DNase I or antiL-DNase II antibodies which determined the resident DNases involved in DNA degradation. According to activity measurements, the highest degradation was obtained in nuclei from E11 retinas but degradation was maximal under acid DNases activating conditions. Moreover, DNA was protected when the experiment was done in the presence of antiL-DNase II. These results indicate that even if other nucleases bearing DNase II activity may exist or if DNA could be degraded by the acidic conditions used, L-DNase II seems responsible for most of DNA degradation in these nuclei. In addition, in spite of the presence of DNase I in retina nuclei at E11, its participation to DNA degradation is not very important since no protection is obtained with anti-DNase I, an antibody that inhibits DNase I activity (Torriglia et al., 1995). This does not rule out completely the participation of DNase I but strongly suggests that other DNases Ca2 -Mg2 -dependent different than DNase I may be involved. From the different Ca2 Mg2-dependent DNases implicated in apoptosis some of them, like DNase g (Shiokawa, Iwamatsu and Tanuma, 1997), for instance, have been fully characterized and their involvement in retina apoptosis is under current investigation. The results obtained for the developing retina are similar to those obtained in the lens (Torriglia et al., 1995) and in other cell models, like the HeLa cells, when apoptosis has been induced by long term culture (Torriglia et al., 1999). In HeLa cells the activation of the L-DNase pathway does not impair the activation of other nucleases at the same time, just like in the developing retina. We may then presume that the L-DNase II apoptotic pathway is activated when cells are not receiving the extracellular signals to survive (Jacobson, Weil and Raff, 1997). However, this is not the only pathway activated. Other apoptotic pathways seem to be important to retina development, such as, the caspasedependent pathways. Actually, the inactivation of caspase 3, via gene inactivation of Apaf-1 generates defects in retina development (Cecconi et al., 1998). However 5 % of these mutants survive and the central nervous system develops normally (Honarpour et al.,
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2000). It is thus possible that different apoptotic pathways are activated in individual cells according to the speci®c signal that triggers apoptosis in the cell. Therefore, several apoptotic pathways may run concurrently to regulate cell number. Acknowledgements We acknowledge Dr Jean Claude Jeanny and Laurent Jonet for the sections, Dr Lisa Oliver for correcting the English manuscript, and Herve Coet for the photographs. This work was supported by INSERM, APEX contract 4X012D and by Retina France.
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