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1 Department of Pediatrics, Section of Respiratory Medicine, Departments of 2 Molecular and Cellular Physiology and 3 Genetics, Boyer Center for Molecular Medicine, and 4 The Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3-13 were subjected to O2 deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ~20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2 deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.
green fluorescent protein-kinesin; cell cycle arrest; O2 deprivation; invertebrates; metaphase; S phase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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RECENTLY, IT HAS BEEN RECOGNIZED that the study of the effect of O2 deprivation is not only important to uncover pathogenetic mechanisms of disease processes, but also to better understand development and cell growth in early life, especially during embryogenesis (10, 30, 31). Most vertebrate species are extremely intolerant of severe or prolonged hypoxic exposure, and O2 deprivation in utero can often result in fetal demise. However, several animals, such as the turtle and brine shrimp, demonstrate imperviousness to lack of O2 (2). We and others previously showed that the fruit fly Drosophila melanogaster embryo or adult is also capable of surviving and recovering from several hours of prolonged hypoxia (13, 35). The understanding of the cellular and molecular mechanisms underlying the response to hypoxia and those at the basis of cell injury and lack thereof in this and other species has been essentially limited.
Of special importance to this work is the fact that, clinically, patients who live for prolonged periods of time with desaturated hemoglobin (such as in congenital heart disease) show stunted growth and development in early life (12) that is not likely related to dietary intake. Similar growth-related problems have also been discovered in high-altitude sojourners (17). Therefore, low O2 conditions may not only lead to injury but may also affect growth rate early in life.
The use of green fluorescent protein (GFP) technology has afforded us the opportunity to visualize molecular and organellar events in real time of living organisms (16, 27). In this report, we describe the use of D. melanogaster embryos with a GFP-tagged kinesin and confocal microscopy to analyze in vivo the effect of low O2 on cell cycle activity and development and viability of the early organism. We report that the exposure of early D. melanogaster embryos to O2 lack induces cell cycle arrest at two major points in the cell cycle, interphase (S phase) and metaphase. This O2 lack appears to inhibit the degradation of cyclin B that may be involved in inducing metaphase arrest. Finally, on reoxygenation, fruit fly embryos recover cell cycle activity and resume their developmental programs, even after 30 min to 2 h of O2 deprivation.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials
5'-bromo-2-deoxyuridine (BrdU), propidium iodide, Shields and Sang M3 insect medium, and the
-tubulin antibody
were obtained from Sigma (St. Louis, MO). The anti-BrdU antibody was
purchased from Becton-Dickinson (San Jose, CA). The antibody to cyclin
B (Rb 271) was kindly provided by David M. Glover (Cambridge
University, Cambridge, UK) (32). All secondary antibodies
and donkey sera were obtained from Jackson ImmunoResearch Laboratories
(West Grove, PA).
Fly Stocks
Initial studies were performed utilizing wild-type flies of the Canton S strain (Bloominfield Center, IN). Flies containing the GFP-kinesin construct Ma+-+-GFP2l2 (II). The heat shock String fly strain [y, Df(w)67; HS-STG3] was provided by Dr. Bruce A. Edgar (4).Egg Collection and Preparation
Fly stocks were maintained in population cages at 25°C under standard conditions. Embryos were collected on apple juice plates supplemented with a small amount of yeast paste. Generally, the first two collections of the day were discarded and subsequent collections were made at 30 min, 1, 2, or 4 h. Embryos were rinsed from the agar plates into a Nytex mesh holder with a solution containing 0.7% NaCl and 0.02% Triton X-100 (TX-100; Sigma). They were then thoroughly rinsed with distilled deionized H2O (DDW). Embryos were then dechorionated in 50% domestic bleach (sodium hypochlorite; Chlorox, Oakland, CA) in DDW for 2 min and again rinsed thoroughly with DDW prior to any experimental manipulation. Twenty-three embryos were analyzed in detail with in vivo confocal imaging.Observing Living Embryos During Normoxia and Hypoxia
Dechorionated GFP-kinesin fly embryos were mounted onto coverslips, according to Schubiger and Edgar (29), and each coverslip was placed into a specially designed perfusion chamber. The embryos were then enclosed within the perfusion chamber with a circular Thermanox plastic coverslip (15-mm diameter; Nalge Nunc International, Naperville, IL), immediately perfused with normoxic insect medium, and viewed with a Bio-Rad MRC-1024 confocal microscope system (Bio-Rad Laboratories, Hercules, CA). Images were captured every 30-40 s from GFP-kinesin embryos for 1 or 2 cell cycles under control, normoxic conditions before switching to an hypoxic insect medium. The hypoxic medium had been previously bubbled with 100% N2 for 2 h before the experiment and was continously bubbled with the same gas throughout the experiment. Measurements of PO2 were made during some experiments, and PO2 was maintained at 10-20 mmHg. Perfusion with the hypoxic solution was maintained for 30 min, 1 h, or 2 h. After the hypoxic period, perfusion with normoxic insect medium was reinitiated, and, on the resumption of cell cycle activity, embryos were observed for an additional one or two cell cycles during reoxygenation.Immunocytochemical and Immunoblot Analysis of the Effect of Hypoxia on Drosophila Embryos
Hypoxia chamber. Dechorionated embryos were placed into a specially designed hypoxia chamber that allowed the manipulation of the embryos while totally preserving the hypoxic environment. Embryos were then subjected to hypoxia for varying periods of time (20, 30, 60, and 120 min). At the end of the hypoxic exposure, embryos were either permeabilized and fixed within the chamber for immunocytochemical analysis or quickly removed and frozen in liquid N2 for immunoblot analyses. The appropriate controls were also run concurrently in room air or bubbled with 21% O2.
BrdU incorporation studies. With cell and life cycles as short as those of the D. melanogaster, the collection of embryos of a similar age is rather tedious. This limitation was circumvented by the use of a heat shock String (stg) fly strain. We utilized a fruit fly strain carrying a heat shock protein 70 promoter-String (cdc25 homolog) construct that permits embryonic cells resting in G2 to be heat-pulsed into an early but well-synchronized mitosis and subsequent S phase. HS-STG3 embryos were collected on 100-mm apple juice plates over 30 min. The HS-STG3 embryos were then aged for 130 min to generate embryos that had completed the first 13 cell cycle divisions and had entered the first G2 gap phase of Drosophila embryogenesis in interphase 14. During the aging process, the embryos were dechorionated and permeabilized with 100% n-octane (Sigma) to permit BrdU incorporation. Two groups of embryos, one to be exposed to hypoxia and the other to serve as a heat shock control, were then placed in an incubator at 37°C for 40 min. A heat shock pulse of this duration induces global stg mRNA and STG protein expression within the embryo and synchronously drives all G2 cells into mitosis within ~5-10 min of returning the embryos to room temperature (RT). Mitosis is generally completed in 10-20 min, and a singular S phase that lasts for 45 min immediately follows this "premature" mitosis. The cells then enter another G2 and remain there for 70-170 min before resuming the normal developmental program (4). Heat-pulsed HS-STG3 embryos were therefore simultaneously exposed to chamber hypoxia, as described above, and incubated in insect medium containing the thymidine analog BrdU (1 mg/ml) for 20-30 min so we could assess DNA-replication activity during O2 deprivation. HS-STG3 embryos that were neither heat shocked nor exposed to hypoxia and those that were heat shocked but maintained in room air served as parallel controls. Embryos were then either permeabilized and fixed within the hypoxia chamber for BrdU immunocytochemistry or removed quickly and frozen in liquid N2 for immunoblotting.
Whole mount immunocytochemistry.
For immunocytochemical and immunoblot studies, we used
cellularized rather than syncytial embryos because we needed to
synchronize the cell cycle stage among embryos. This necessitated the
use of HS-STG3 embryos, which relies on aging embryos until
interphase 14. Whole mount immunocytochemistry of
embryos was performed essentially as described by Fehon et al.
(9). Embryos were permeabilized by shaking in 100%
heptane (Sigma) for 30 s before being transferred to a solution of
50% heptane and 2% paraformaldehyde (for BrdU) or 37% formaldehyde
(for -tubulin) in PBS for 30 min. The vitelline membrane was removed
by incubation in methanol, and, if the embryos were not immediately
processed for immunocytochemistry, they were stored at 
20°C.
Rehydrated embryos were rinsed three times for 5 min each in PBS, 0.2%
BSA, and 0.1% TX-100 (PBT). They were then incubated in 10% normal
donkey serum (NDS) and 0.3% TX-100 in PBT for at least 1 h at RT.
Embryos were incubated in primary antibody in 10% NDS and 0.3% TX-100
in PBT overnight at 4°C. After being rinsed in PBT, embryos were
incubated in the secondary antibody, also in 10% NDS, 0.3% TX-100,
and PBT for 2 h at RT. Embryos were finally rinsed twice 5 min
each with PBT and once for 5 min in PBS before being mounted onto
slides in Vectashield (Vector, Burlingame, CA) and placed on a
coverslip. DNA staining performed in conjunction with antibody
staining involved incubation with 200 µg/ml RNAse A (Sigma) during
the primary antibody step as well as in the three rinses in PBT after
the secondary antibody step. Embryos were then incubated in 10 µg/ml
propidium iodide in PBT for 3-5 min and thereafter rinsed twice, 5 min each, in PBT and once for 5 min in PBS prior to mounting.
Immunoblotting. For Western blotting, dechorionated embryos were homogenized in Laemmli sample buffer (2% SDS, 10% glycerol, and 67 mM Tris, pH 6.7) containing protease inhibitors. Embryo samples were assayed for protein content (BCA Assay, Pierce Chemical, Rockford, IL), and 10 µg of total protein was loaded per lane for SDS-PAGE and subsequent transfer to polyvinylidene fluoride membranes (Immobilin-P, Millipore, Bedford, MA). Proteins were detected with secondary antibodies conjugated with horseradish peroxidase and the enhanced chemiluminescence system (Amersham, Little Chalfont, UK) as described previously (14). Scanning densitometry of immunoblot films was performed on a Personal Densitometer SI scanner (Molecular Dynamics, Sunnyvale, CA) and analyzed with ImageQuaNT image- analysis software (Molecular Dynamics).
Data Analysis
Data are reported as means ± SE.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Live Confocal Imaging of Early Embryogenesis With and Without O2
In vivo confocal imaging of cell cycle activity in Drosophila
embryos under control, normoxic conditions.
Embryos undergoing nuclear division cycles 3-13 were imaged on the
confocal microscope while being perfused with Shields & Sang M3 insect
medium. After an equilibration period of 10-15 min, GFP
fluorescence is seen along the extent of the spindle fiber apparatus,
inclusive of astral fibers at particular stages during the cell cycle,
and is localized to centrosomes throughout the cell cycle (Fig.
1). The first evidence of mitotic
activity is the appearance of relatively amorphous spindle fiber
apparatuses extending between the two polar centrosomes (prometaphase
stage of mitosis, as seen in Fig.
2G). Within the subsequent few
minutes, GFP fluorescence increases markedly and distinct spindle fiber bundles separated by a dark central portion (paired sister chromatids) become evident, as cells enter metaphase proper (Fig. 1A).
Embryonic cells remain in metaphase for ~2.5 min but, shortly after,
the spindle fibers begin to separate and move to opposite poles of the
energid (nuclei and associated cytoplasm) (1) at a time when the astral fibers surrounding the centrosomes become apparent (metaphase to anaphase, Fig. 1B). As the spindle fibers
continue to migrate outward during the anaphase to telophase transition (Fig. 1C), spindle fibers become less ovoidal and more
rectangular. During telophase and cytokinesis (Fig. 1, D and
E), the two new daughter nuclei can also be detected as
darkened circles outlined by a halo of fluorescence. The centrosomes of
the daughter energids, which are initially located on one side of the
cell, then replicate and can be seen to migrate along the perimeter of
the two newly forming daughter cells to assume polar positions around
the nucleus (Fig. 1F). About 12 min from the start of
metaphase, most or all of the energids of the embryo have entered S
phase (Fig. 1F), where the dark circles represent the new
daughter nuclei. The manipulation of the embryos under these
experimental conditions did not have any deleterious effects on
subsequent development.
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Effect of hypoxia on cell cycle activity of GFP-kinesin embryos. In these experiments, hypoxia exposure of 30, 60, or 120 min was initiated at a variety of points during the cell cycle. Arrest was achieved both with 100% N2 only and also with solutions bubbled with 5%O2 in 95% N2. Two major arrest stages resulted from these in vivo perfusion hypoxia experiments.
INTERPHASE ARREST. Embryos exposed to hypoxia from the period just after the metaphase stage of mitosis, e.g., during the metaphase-to-anaphase transition (Fig. 2A) to very early in S phase, continue to progress (Fig. 2, B-D), and do not arrest until S phase (Fig. 2E). At this phase, embryos appeared to have arrested and do not progress for the duration of the hypoxic exposure. If the hypoxia is maintained for 1 h or longer, at ~45-55 min of hypoxic exposure, fluorescent signals generated by the centrosomes generally dissipate to background levels. As can be seen in Fig. 2, the exposure of these embryos to hypoxia does not seem to have any deleterious effects on spindle fiber morphology or movement. Indeed, reoxygenation of embryos resulted in the resumption of cell cycle activity, within ~20 min of the return to normoxia (Fig. 2G). The first evidence of recovery during reoxygenation is usually the marked increase in GFP-fluorescence of the centrosomes within ~5 min (Fig. 2F). Interphase-arrested embryos resume mitotic activity, as evidenced by the appearance of centrosomes followed by amorphous spindle fibers overlying the nuclei during prometaphase (Fig. 2G). Subsequently, embryonic cells divide, as can be seen in Fig. 2H. METAPHASE ARREST. Embryos exposed to hypoxia very late in S phase, i.e., just after DNA replication but before metaphase proper, arrest in metaphase (Fig. 3). Figure 3A shows an embryo whose energids appear to be in either late prophase or prometaphase. Hypoxia was initiated just as the energids were entering metaphase (Fig. 3B). Unlike the embryo depicted in Fig. 2 (S phase arrest), this embryo arrested in metaphase (Fig. 3, C and D) without migrating through any other stage of mitosis. Two obvious phenomena occurred during a metaphase arrest. 1) The centrosomes lose their GFP-fluorescence intensity over time, as in the previous embryo. Centrosomes and astral fibers, visible in Fig. 3, B and C, are no longer apparent in Fig. 3D. 2) The spindle fiber apparatus undergoes specific and consistent morphological changes during hypoxia. The spindle fiber apparatus assumes a more ellipsoid (thinner) and condensed configuration and demonstrates some loss of fluorescent intensity during hypoxia (Fig. 3, B-D). The energids remain arrested in metaphase for the duration (up to 2 h tested) of hypoxia. On reoxygenation, the first recordable event is the reappearance of the centrosomes, as occurs after an S phase arrest (Fig. 3E). Thereafter, the spindle fiber apparatus demonstrates a marked increase in fluorescent intensity and a widening of its structure. The spindle fiber apparatus then resumes its normal metaphase conformation, and the cells resume cycling, again within ~20 min of reoxygenation (Fig. 3F). It is noteworthy that embryos exposed to hypoxia before metaphase (e.g., during prometaphase) that arrest at metaphase do so in a very short period of time, i.e., 1-2 min.
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Determination of Hypoxia-Induced Cell Cycle Arrest Points
Effect of hypoxia on the distribution and expression of -tubulin
and propidium iodide.
interphase arrest.
Embryos examined after 2 h of hypoxia demonstrated an arrest at
either metaphase or S phase. A gastrula-stage embryo that appears to be
arrested in S phase is depicted in Fig.
4A. The chromatin material is
stained red by propidium iodide, and the -tubulin antibody stains
membranous material green. All of the cells in the gastrula-stage
embryo seem to have arrested at the same stage (S phase) of the cell
cycle. The chromatin material has a "prophase" configuration, in
that the chromatin is condensed and somewhat circular. The -tubulin
stains the plasma membrane and intracellular cytoskeletal elements.
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-tubulin antibody, is remarkably similar in conformation to that
observed during metaphase arrest in the in vivo GFP experiments. The
spindle apparatus appears thinner and elliptical.
Therefore, both experiments in live and fixed tissues demonstrate that
arrests in the cell cycle mediated by hypoxia occur at metaphase or
during S phase. The early embryo has no G1 and G2 gap phases; however,
the above experiments did not indicate whether hypoxia could interfere
with DNA synthesis.
Effect of hypoxia on the incorporation of BrdU. In assessing both the live and fixed tissues, we determined that there were essentially two hypoxia-induced arrest points, at the metaphase-to-anaphase transition and at S phase. However, it was not clear from these experiments where embryos arrested, vis-à-vis DNA replication, when they were exposed to O2 lack past metaphase or anaphase. To address this issue, we exposed embryos to hypoxia in a specially designed chamber that permitted the permeabilization and fixation of embryos within the hypoxic environment. To determine the exact time that embryos arrested during S phase, we found it useful to utilize a fly strain that, on entering into the first gap phase in embryogenesis (G2 of S phase 14), could be induced to enter mitosis synchronously. This protocol, which is based on the use of heat shock String overexpression (see MATERIALS AND METHODS), provides gastrula-stage embryos whose cells enter mitosis and the subsequent S and G2 phases simultaneously.
Embryos were collected and allowed to age for 130 min, which would bring them to the G2 phase of interphase 14. They were then heat shocked for 40 min at 37°C. Control embryos that were not subjected to either heat shock or hypoxia demonstrated the incorporation of BrdU into nuclei within discrete mitotic regions (Fig. 5A). BrdU, in green, was detected along the ventral surface, where actively dividing neural precursors reside, and within nuclei of the mitotic domains of the gut region of this gastrula embryo, whereas chromatin material is stained red by propidium iodide. Embryos that were subjected to 40 min of heat shock but no hypoxia (heat shock control) gave evidence of more global BrdU incorporation, indicating that heat shock-induced String expression had driven most of the cells of this gastrula embryo into a premature mitosis and associated S phase (Fig. 5B). Embryos that were exposed to hypoxia within 10-15 min after heat shock showed absolutely no BrdU incorporation (Fig. 5C), indicating that hypoxia prevented these cells from initiating DNA replication. This study verified that hypoxia arrests embryos in interphase before DNA replication.
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Effect of hypoxia on cyclin B expression.
Again taking advantage of the heat shock String protocol, we examined
the expression of cyclin B during hypoxia (n = 2).
Cyclin B protein expression levels were normalized to -tubulin
protein expression levels and are presented in arbitrary units (AU)
(Fig. 6). Control embryos that were heat
shocked (Fig. 6, lane 2) had similar levels of cyclin B
expression (0.64 ± 0.18 AU) compared with control embryos that
were not heat shocked (0.84 ± 0.37 AU; Fig. 6, lane
1), indicating that the heat shock protocol did not have a
significant effect on cyclin B expression in these S phase embryos.
Levels of cyclin B expression in embryos that were heat shocked and
exposed to hypoxia 4 min after the heat pulse (0.75 ± 0.38 AU;
Fig. 6, lane 3) were similar to both non-heat-shocked and
heat-shocked controls, indicating that these embryos were arrested in
metaphase with undegraded cyclin B. On the other hand, heat-shocked
embryos that were exposed to hypoxia 12 min after the heat pulse (Fig.
6, lane 4) showed lower levels of cyclin B expression
(0.38 ± 0.11 AU) than the controls and the 4-min hypoxia group,
indicating that they had passed through metaphase and had arrested in S
phase with degraded cyclin B.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Within the cell cycle of both prokaryotes and eukaryotes are a number of transition states that can be interrupted if certain requirements have not been met or if the regulation of a number of cell cycle regulatory proteins has been altered (5, 15, 26, 28). In this study, we have identified cell cycle checkpoints that are responsive to O2 levels. Indeed, our in vivo analysis of fruit fly embryos containing a GFP-kinesin construct revealed that hypoxia induces cell cycle arrest at either S phase (before DNA duplication) or metaphase. These arrest points are dependent on the stage of the cell cycle when the embryo is exposed to O2 deprivation.
Although there have been two detailed studies on the effect of hypoxia on fly embryos, our study has differed in a number of respects (10, 35). First, this is the first study showing, in live embryos, online cell division of syncytial and cellularized embryos that arrest in very low O2 conditions. Second, although one of the other two studies (10) shows an arrest in the developmental program of the embryos during mitosis, the study did not show any detail regarding the relationship between the start of hypoxia and the stage at which the embryo arrested. Third, unlike the previous two studies, we describe the alterations in the shape of the spindle fibers when embryos are arrested in metaphase and the reversibility of this process. Fourth, unlike our study, in neither of the other studies did the authors investigate cell cycle checkpoint proteins.
Specificity of Hypoxia-Induced Cell Cycle Response
Depriving D. melanogaster embryos of O2 resulted in a consistent and reproducible cell cycle arrest at either S phase or metaphase. Reoxygenation led to the resumption of cell cycle activity within ~20 min, again in a relatively stereotypical fashion. In this study, we have been able to demonstrate that fruit fly embryos can maintain the arrested state for up to 2 h and resume cell cycle activity on reoxygenation. This can be repeated several times even within the same embryo. It is reasonable then to assume that the presence or absence of O2 has specific, reproducible effects on cell cycle activity. These data support the idea that O2 deprivation has a unique mechanism of action on the cell cycle in fruit fly embryos. Furthermore, the rapidity of the induced arrest suggests that there is a direct effect of decreased O2 levels on the cell cycle machinery rather than an effect on energy stores that would generally take a longer time.Possible Mechanisms of Action of Hypoxia-Induced Arrest
Hypoxia-induced S phase arrest, based on in vivo confocal and BrdU incorporation studies, seems to occur before DNA replication. This indicates that DNA replication is sensitive to the availability of O2 and that at least one or more components of the DNA-replication checkpoint machinery may be hypoxia responsive. Two categories of DNA checkpoints have been described: 1) DNA replication checkpoint and 2) DNA damage checkpoint (8, 33). Because these embryos recover cell cycle activity rather quickly during reoxygenation, it is more likely that the DNA-replication checkpoint is invoked during hypoxia to produce an S phase arrest. A number of DNA-replication checkpoint proteins have been implicated, and some could be important in transducing the hypoxia signal. These would include cyclin-dependent kinases 1 (cdc2), 2, 4, and 6; cyclins A, D, and E; the retinoblastoma protein; and cyclin-dependent kinase inhibitors (11, 21, 22).A block of the cell cycle at metaphase can occur if the spindle
apparatus is improperly assembled or if sister chromatid separation is
prevented (5, 28). In our study, hypoxia-exposed embryos specifically arrested at this mitotic spindle checkpoint, as
demonstrated in the in vivo confocal and -tubulin immunocytochemical
studies. Therefore, a release from metaphase was inhibited by the
hypoxic exposure. The metaphase-to-anaphase transition requires the
activation and inactivation of several cell cycle regulatory proteins
(3), and the release from metaphase requires the
degradation of cyclin B and the inactivation of cdc2 by
ubiquitin-dependent proteolytic activity of the anaphase promoting
complex (APC) (19, 25, 34). Additionally, proteins such as
the securins and separins that control sister chromatid cohesion and
separation, as well as a block on the APC, could therefore be involved
in the hypoxia-induced metaphase arrest (18, 20). Because
we have demonstrated that cyclin B is not degraded during a
hypoxia-induced metaphase arrest, it can therefore be hypothesized that
O2 deprivation imposes a block on cyclin B degradation
either by maintaining the inactivated state of the APC or preventing
activation of the APC. However, as noted in Fig. 6, cyclin B does not
appear to be completely degraded after metaphase. It has been reported
that cyclins A and B are not completely degraded after metaphase in
syncytial (early) embryos (23) and that a 37°C heat
shock may also delay the degradation of cyclins A and B in blastoderm
(later stage) embryos (24).
Significance
The hypoxia-induced arrest in embryos may have a beneficial value, possibly related to survival of the organism. Besides saving on energy requirements, an arrest during S phase before DNA replication may function to safeguard against DNA-replication errors. Similarly, an arrest during metaphase may serve to protect the cell from errors associated with abnormal spindle morphology and migration, improper chromatin alignment on the spindle apparatus, and abnormal or dysfunctional sister chromatid separation during hypoxia (15, 26). What is still a mystery, however, is the molecular basis for the lack of apparent injury to the organism during resumption of growth after hours, or even days and weeks, of hypoxia/anoxia and developmental arrest as has been described for Drosophila embryos exposed to chronic hypoxia for up to 8 days (35) and brine shrimp embryos maintained in complete N2 for up to 4 yr (2). Furthermore, because in utero and early postnatal growth is also profoundly affected by O2 deprivation such as at high altitude, it is very likely that mechanisms similar to those described in this study are also present in other organisms, including mammals. Indeed, it has become clear in the past decade that organisms such as Drosophila and Caenorhabditis elegans share with mammals and even primates the same complements of genes that shape development, growth, and response to stress. This concept has rendered these genetic models very attractive since findings in these models may be generalizable.Perspectives
That cell cycle and cell division are affected during low O2 states does not need to be limited to this species. As mentioned above, the shrimp embryo is also affected in a major way. There is indeed evidence that growth in mammals, including humans, is sensitive to low O2 states. Experiments from our laboratory in the past few years have always demonstrated that rodents exposed to chronic or intermittent hypoxia in early life are smaller. In utero growth and postnatal development can be retarded, and lessons from young patients with cyanotic congenital heart disease have taught us that growth rate can be impaired in these patients. Whether healing from injury and repair mechanisms is also affected by hypoxia remains to be seen and needs additional experimentation.| |
ACKNOWLEDGEMENTS |
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The authors are grateful to Xiaolan Fei, Ralph Garcia, and Phillipe Male for providing superb technical assistance with this project and to Drs. Michael Nathanson and Stewart Frankel for pertinent discussions related to confocal microscopy and genetics.
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FOOTNOTES |
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T. Xu was supported by National Cancer Institute Grant CA-69408, the Pew Scholar Award, and Howard Hughes Medical Institute; G. G. Haddad was supported by National Institutes of Health Grants T32-HL-07778, NS-35918, NS-37756, and PO1-HD-32573.
Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics, Section of Respiratory Medicine, Yale Univ. School of Medicine, 333 Cedar St., FMP 506, New Haven, CT 06520 (E-mail: gabriel.haddad{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 November 2000; accepted in final form 10 January 2001.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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