Department of Ophthalmology and Visual Sciences, Washington University Medical School, St. Louis, Missouri 63110-1093
During development, the lens of the eye becomes transparent, in part because of the elimination of
nuclei and other organelles from the central lens fiber
cells by an apoptotic-like mechanism. Using confocal
microscopy we showed that, at the border of the organelle-free zone (OFZ), fiber cell nuclei became suddenly irregular in shape, with marginalized chromatin.
Subsequently, holes appeared in the nuclear envelope
and underlying laminae, and the nuclei collapsed into
condensed, spherical structures. Nuclear remnants, containing DNA, histones, lamin B2, and fragments of
nuclear membrane, were detected deep in the OFZ.
We used in situ electrophoresis to demonstrate that
fragmented DNA was present only in cells bordering the OFZ. Confocal microscopy of terminal deoxynucleotidyl transferase (TdT)-labeled lens slices confirmed
that DNA fragmentation was a relatively late event in
fiber differentiation, occurring after the loss of the nuclear membrane. DNA fragments with 3-OH or 3
-PO4
ends were not observed elsewhere in the lens under
normal conditions, although they could be produced by
pretreatment with DNase I or micrococcal nuclease, respectively. Dual labeling with TdT and an antibody
against protein disulfide isomerase, an ER-resident protein, revealed a distinct spatial and temporal gap between the disappearance of ER and nuclear membranes and the onset of DNA degradation. Thus, fiber
cell chromatin disassembly differs significantly from
classical apoptosis, in both the sequence of events and
the time course of the process. The fact that DNA degradation occurs only after the disappearance of mitochondrial, ER, and nuclear membranes suggests that
damage to intracellular membranes may be an initiating event in nuclear breakdown.
The lens of the eye is a transparent cellular structure
that focuses light on the retina. The bulk of the lens
consists of concentric layers of fiber cells that are
formed throughout life by the differentiation of cells at the
equatorial margin of the lens epithelium (see Fig. 1). Fiber
cell differentiation is characterized by cellular elongation,
the synthesis of certain crystallin proteins, and the degradation of all membrane-bound organelles, including nuclei
(Piatigorsky, 1981
During development, optical clarity is ensured by the
degradation of intracellular organelles in fiber cells near
the center of the lens (i.e., those cells directly in the light
path). The mechanism by which organelles are disassembled is not known, but recent work has demonstrated that
this process is under strict temporal and spatial control
and that, in any given cell, the endoplasmic reticulum, mitochondria, and nuclei are rapidly and synchronously degraded (Bassnett, 1992 In the embryonic lens, organelles are present initially
throughout the volume of the tissue, but, beginning on embryonic day 12 (E12)1 in the chicken, they are eliminated
from cells in the center of the lens (Bassnett and Beebe,
1992 Because both processes involve the systematic degradation of chromatin, several authors have pointed out the
parallels between lens fiber cell denucleation and classical
apoptosis (Chaudun et al., 1994 It has been known since the turn of the century that differentiation of lens fiber cells involves the loss of cell nuclei (Rabl, 1899 Although there is a general consensus on the morphological aspects of lens cell denucleation, there is considerable disagreement regarding the underlying biochemical
mechanism(s) and, importantly, the timing of the process.
In a series of experiments in the early 1970s, the then
newly discovered enzyme terminal deoxynucleotidyl transferase (TdT) was used to radiolabel the nuclei of sectioned embryonic chicken lenses (Modak and Bollum, 1970 However, in marked contrast to the work described
above, recent studies have been unable to detect the presence of DNA strand breaks in differentiating chicken embryo fiber cells (Chaudun et al., 1994 Animals
Fertilized White Leghorn chicken eggs were obtained from Truslow
Farms (Chestertown, MD) and incubated at 38°C in a forced-draft incubator. Embryonic lenses, with their attached vitreous humor, were removed
through an incision in the posterior globe.
Preparation of Lens Slices
Fixed lenses were embedded in 4% agar/PBS and sectioned in the midsagittal plane using a Vibratome (model 3000; Technical Products International, Inc., St. Louis, MO) as previously described (Bassnett, 1995 DNA and Membrane Staining
In some preparations, it was necessary to colocalize DNA and cellular
membranes. This was done by washing fixed lens slices in TBS (10 mM
Tris, 150 mM NaCl, pH 7.4) for 5 min and then incubating the slices for 40 min in TBS containing 0.005% Triton X-100, 1% BSA, 1 µM SYTO 17 (Molecular Probes, Eugene, OR), and 1 µg/ml 3,3 Immunofluorescence
E18 lenses were fixed for 1 h in 4% paraformaldehyde/PBS, pH 7.4, and
sliced at 200 µm. Slices were permeabilized with 0.1% Triton X-100 for 30 min at room temperature and incubated in blocking solution (10% normal
goat serum/1% BSA/PBS) for 1 h. Slices were incubated overnight at 4°C
in primary antibody diluted 1:500 in blocking solution. After a 1-h wash in
PBS, slices were incubated for 2 h in fluorescein-conjugated goat anti-
mouse IgG antibody (Jackson ImmunoResearch Labs., West Grove, PA)
diluted 1:500 in blocking solution. Slices were then washed for 1 h in PBS,
mounted, and viewed.
To visualize the fate of histone proteins during fiber differentiation, we
used a monoclonal antibody that recognizes an epitope present in histone
H1, H2A, H2B, H3, and H4 (clone H11-4; Boehringer Mannheim Corp.,
Indianapolis, IN). To visualize the distribution of the ER in lens slices, we
used a monoclonal antibody (clone ID3; StressGen Biotechnologies
Corp., Sidney, BC, Canada) against protein disulfide isomerase (PDI), an
ER-resident protein, as described previously (Bassnett, 1995 In Situ TdT Labeling
Lenses were fixed in 4% paraformaldehyde/PBS, pH 7.4, for 1 h at room
temperature. Lens slices were permeabilized for 30 min in 0.1% Triton
X-100/PBS at room temperature and washed for 20 min in PBS. The TdT
labeling reaction was performed using an in situ cell death detection kit
(Boehringer Mannheim Corp.) according to the manufacturer's instructions. In this assay, a homopolymeric tail of fluorescein-labeled nucleotides is incorporated at the 3 Permeabilized slices were pretreated with DNase I (Boehringer Mannheim Corp., 50 U/ml in 10 mM Tris, pH 7.6) for 30 min at 37°C, as a positive control. DNase I cleaves DNA into fragments with 3 In Situ Electrophoresis
The location of nuclei with degraded DNA was visualized by an in situ
electrophoretic technique under alkaline conditions. This method was
modified from that used previously to measure DNA damage in single
cells (Ostling and Johanson, 1984 Confocal Microscopy
Slices were viewed using a confocal microscope equipped with an Argon/
Krypton laser (model LSM 410; Carl Zeiss Inc., Thornwood, NY).
Cut open along the optic axis, an E15 embryonic chicken
lens has the cellular organization shown in diagrammatic
form in Fig. 1. Confocal microscopic images of the changes
in nuclear organization in cells near the border of the OFZ
are shown in Fig. 2. Lens slices were stained with both
DiOC6 and SYTO 17, enabling the simultaneous visualization of the fiber cell membrane systems (Fig. 2, B, E, H,
and K) and DNA (Fig. 2, C, F, I, and L), respectively, in
addition to differential interference contrast (DIC) images (Fig. 2, A, D, G, and J). In the outer cortex of the lens, the leaf-shaped fiber cell nuclei were the only prominent subcellular feature of the DIC images (Fig. 2 A). The corresponding confocal image obtained with DiOC6 (Fig. 2 C)
showed labeling of the fiber cell membrane systems, including the plasma membrane and the nuclear envelope.
The distribution of DNA was visualized simultaneously using SYTO 17. The fiber cell nuclei were evenly stained by
SYTO 17, with prominent nucleoli. In fiber cells near the
border of the OFZ, a change in the organization of the nuclei was apparent (Fig. 2, D-F). In the space of a few cell layers, nuclei passed through distinct stages. In the first stage,
the leaf-shaped nuclei characteristic of the outer cell layers
became irregular in shape with an undulating nuclear membrane. At this point, the DNA staining was uneven, with
marginalization of the chromatin. Subsequently, the nuclei
collapsed into small spherical structures that were visible
under DIC. Although the collapsed nuclei were intensely stained by the SYTO 17, the chromatin now appeared "naked" (i.e., no longer contained within a nuclear membrane). The last nucleus with an intact nuclear membrane
is indicated by the arrow in Fig. 2 E. Fiber cells containing
collapsed nuclei extend for 100-200 µm into the OFZ.
Even in cells at the very center of the lens, small, positively
stained nuclear remnants could be discerned (Fig. 2 L) in
the otherwise featureless fiber cell cytoplasm.
Confocal microscopy and immunofluorescence were used
to visualize the fate of two nuclear components, lamin B2
(Fig. 3) and histone proteins (Fig. 4), in fiber cells near the
border of the OFZ. Lamin B2 is a member of a family of
intermediate filament proteins that together form the
karyoskeleton and, as such, are usually distributed immediately beneath the nuclear membrane. In the superficial
cortex of the lens, the lamin B2 immunofluorescence specifically delineates the fiber cell nuclear membrane (Fig. 3 A). In these experiments, omission of the primary antibody, or its substitution by ascites fluid, resulted in complete absence of nuclear fluorescence, demonstrating the
specificity of the antibody (data not shown). At the border
of the OFZ, the nuclei collapsed and lamin B2-positive
nuclear debris persisted for hundreds of cell widths into
the OFZ (Fig. 3 B). Immediately before nuclear collapse
(Fig. 3 B, arrow), lamin B2 immunofluorescence became uneven and the nuclear profile was irregular (Fig. 3 C).
Two nuclei in the early stages of nuclear collapse are indicated by arrows in Fig. 3 C. An adjacent nucleus, with a
relatively intact lamina, is indicated by an arrowhead.
Soon after the collapse of the nuclei (Fig. 3, B, arrowhead), rents were evident in the nuclear lamina (Fig. 3, D).
The histones are the major protein components of the
chromatin. The nuclei of epithelial cells and superficial fiber cells were readily labeled by the histone antibody (Fig.
4, A and B). As the denucleation process commenced, at
the border of the OFZ, the change in the pattern of nuclear histone immunofluorescence paralleled that of the
nuclear DNA (compare Fig. 4 D with Fig. 2 F). Within a
few cell widths, the histone immunofluorescence became
heterogeneous and marginalized, and it ultimately collapsed into brightly stained residual structures (Fig. 4, C
and D).
As the karyoskeleton was disassembled at the border of
the OFZ, the integrity of the nuclear envelope was also
lost. Once the nuclei collapsed, only ragged fragments of
membrane remained, adhering to the naked chromatin
(Fig. 5). The condition of nuclear DNA was also visualized
during various stages of fiber cell differentiation using an
in situ TdT assay. In this assay, a tail of fluorescein-labeled dUTP molecules is incorporated at 3
It has been suggested that a DNase II-like enzyme may
play a role in lens nuclear degradation (Chaudun et al., 1994 From studies such as those shown in Fig. 6 A, it appeared that positively labeled nuclei (i.e., those containing
degraded DNA) were only detected some distance within
the OFZ. If this were true, it would suggest that extensive
DNA cleavage occurs some time after the disappearance of
mitochondria, endoplasmic reticulum, and other organelles.
To verify that this is the correct sequence of events, we
performed immunofluorescence labeling of PDI (an ER
marker) on E19 lens slices that had previously been labeled
with TdT. In lens slices labeled with both TdT and antiPDI, a clear gap was revealed between the border of the
OFZ (delineated by the PDI immunofluorescence) and
the first TdT-positive nuclei (Fig. 7 A). At E19, cells that
were unstained by either treatment formed a region ~90
µm wide. We also noted an interesting developmental sequence in slices labeled with both TdT and anti-PDI. At
E12, the OFZ was already clearly delineated, as described
previously (Bassnett and Beebe, 1992
In contrast to the fiber cells, all the epithelial cells contained abundant ER (as evidenced by strong PDI immunofluorescence), including those rare cells with TdT-labeled
nuclei. Fig. 7 B shows a merged confocal micrograph of
the central region of an E9 lens epithelium. The embryonic lens epithelium contained a few scattered apoptotic
cells, and these were strongly labeled by the TdT assay. The labeled nuclei had a characteristic condensed morphology with small, positively labeled apoptotic bodies often found in close association (Fig. 7 B, arrows).
We also used a novel nonenzymatic technique for visualizing the condition of the chromatin during fiber cell differentiation. In our in situ electrophoresis assay, a lens
slice was embedded in agarose, permeabilized, and oriented perpendicular to a weak electric field. Fragments of
DNA that were sufficiently small to be electrophoretically
mobile were driven from the lens cells into the agarose,
where they were stained with ethidium bromide. A diagram showing the orientation of the lens slice and the expected appearance of the tissue under the confocal microscope is shown in Fig. 8. The optical-sectioning capability
of the confocal microscope enabled us to position the focal
plane at the level of the fiber cell nuclei. With this optical
arrangement, the OFZ appeared as a dark gap between
two arms of strongly fluorescent nuclei (Fig. 9 A). If the
lens slice was pretreated with DNase I before electrophoresis, two diffuse clouds of ethidium-stained material
were observed emanating from the fiber cells (Fig. 9 B). The
clouds of DNA appeared to come from all the annular pad
and fiber cells. In contrast, in non-DNase I-treated slices,
ethidium-stained material was only observed emanating from
cells at the immediate border of the OFZ (Fig. 9, C and D).
We assume that, in each case, the ethidium-stained material emanating from the lens slices represents low-molecular weight DNA because cellular RNA (which would also
be weakly stained by ethidium bromide) is degraded under the extremely alkaline conditions of the assay.
The programmed loss of lens cell organelles is one of the
most characteristic features of lens fiber cell differentiation and also one of the least well understood. Modak and
Bollum suggested that lens cell denucleation is a gradual
process characterized by the steady accumulation of DNA
damage (1970, 1971, 1972). Such damage might result from
the impairment of DNA repair mechanisms in the lens fiber cells (Counis et al., 1981 In previous studies, we noted that the loss of organelles
from lens fiber cells occurs over a time course of a few
hours and within the space of a few cells (Bassnett and
Beebe, 1992 In the present experiments, we used CIAP treatment to
determine whether 3 Lens nuclear breakdown is associated with the appearance of 3 Lens transparency depends on the maintenance of a
highly regular optically homogeneous cytoplasm. If fibers
near the center of the lens were to undergo classical apoptosis, the transparency of the tissue would be compromised, and, presumably, a nuclear cataract would result.
Perhaps the denucleation process in the lens has evolved
in response to twin evolutionary pressures; the need to
eliminate light-scattering organelles without disturbing the
optical properties of the tissue.
The enzyme(s) responsible for digesting nuclear DNA
in cells at the border of the OFZ remains to be identified.
The apparent rapidity of the denucleation process suggests
that a failure of DNA repair mechanisms (Counis et al.,
1981 From the data reported here and previous studies
(Bassnett and Beebe, 1992). Because there is no cell turnover in
the lens, all cells are retained within the tissue, those nearest the center being the oldest and those nearest the surface being the youngest.
Fig. 1.
Diagram of a midsagittal slice of a chicken lens
at E15. The lens is bounded
by an acellular collagenous
capsule. An epithelial monolayer covers the anterior surface of the lens and thickens at the periphery to form the
annular pad. The bulk of the
lens consists of concentric
layers of highly elongated
lens fiber cells derived from
the edges of the epithelium.
Primary fiber cells, formed
early in development, are situated in the center of the lens. The rest of the fiber
mass is composed of secondary
fiber cells, formed throughout life by the differentiation
of epithelial cells at the lens
equator. The tips of secondary fiber cells make contact
with fibers from the opposite
hemisphere of the lens at the
sutures. The outer fiber cells
contain a normal complement of organelles, including nuclei. However, organelles are absent from cells in the center of the lens, giving rise to the OFZ. The
OFZ increases steadily in size throughout embryonic development (small arrows).
[View Larger Version of this Image (43K GIF file)]
, 1995
; Bassnett and Beebe, 1992
).
). This results in the formation of an organelle-free
zone (OFZ; Fig. 1). Once formed, the OFZ expands in diameter at a rate of 80 µm/d during embryonic development (Bassnett and Beebe, 1992
), engulfing ~10,000 cells
per day (Bassnett, 1995
). In adult lenses, those organelles
that remain are concentrated in the periphery of the lens,
lying largely in the shadow of the iris (Bassnett, 1992
). In
certain pathological conditions, however, organelles persist in the central fiber cells. For example, the failure of fiber cells to properly degrade their nuclei is a common feature of human congenital cataract (Zimmerman and Font,
1966
).
; Arruti et al., 1995
; Pan
and Griep, 1995
; Torriglia et al., 1995
). Apoptosis was first
described by Kerr et al. (1972)
as a morphologically distinct form of cell death that involves (among other things)
cell shrinkage, membrane blebbing, and chromatin condensation. One characteristic early feature of apoptotic cell death is internucleosomal DNA cleavage (Wyllie, 1980
),
which has been variously ascribed to the activity of DNase I
(Peitsch et al., 1993
), DNase II (Barry and Eastman, 1993
),
and NUC 18 (Montague et al., 1994
). Interestingly, low-
molecular weight fractions of DNA have been isolated from
anucleate fiber cells in the center of the lens (Appleby and
Modak, 1977
). In fact, the lens was one of the first tissues
in which such "DNA ladders" were observed. However,
an obvious difference between apoptosis and lens fiber cell
denucleation is that, in the latter, the cells are not phagocytosed by their neighbors, and the anucleate fiber cells
persist throughout the life of the organism. One of the purposes of the present study was to determine the extent to
which lens fiber cell denucleation resembles classical apoptosis in its time course and in its biochemical and morphological characteristics.
), and, consequently, this aspect of lens fiber differentiation has received the most attention. The
morphological changes that precede denucleation have
been described in detail for a number of species (see for
example Modak and Perdue, 1970
; Kuwabara and Imaizumi, 1974
; Sanwal et al., 1986
; Vrensen et al., 1991
). During fiber cell maturation, the cell nuclei undergo striking
changes in shape, accompanied by progressive alterations
in chromatin structure and eventual loss of integrity of the
nuclear membrane and infiltration of cytosolic proteins
(Sandilands et al., 1995
).
, 1971
,
1972
). TdT catalyzes the addition of dNTPs to the 3
-hydroxyl
(3
-OH) termini of DNA molecules in a template-independent fashion. The fact that fiber cells contained initiation sites for the addition of radiolabeled nucleotides was
taken as evidence for the presence of significant numbers
of single-strand breaks in the DNA. Quantitative analysis
showed that the nuclei of fiber cells in the deep cortex of
the lens were more heavily labeled than in the superficial cells, suggesting that DNA damage (in the form of free
3
-OH ends) accumulated steadily during fiber cell differentiation. Taken together, these studies support the notion
that fiber cell denucleation is a gradual process, beginning
in the superficial cells and characterized by the steady accumulation of single-strand breaks in DNA. According to
this view, the DNA is ultimately cleaved into small, nucleosome-sized fragments, either by extensive overlapping
single-strand scissions or direct double-strand breaks.
) or in mouse lenses
(Fromm et al., 1994
; Morgenbesser et al., 1994
; Pan and
Griep, 1994
; Chow et al., 1995
; Robinson et al., 1995
), and
DNase I transcripts are not detectable in the lens by reverse transcriptase-PCR (Hess and Fitzgerald, 1996
). The
involvement of a DNase II-like enzyme (which generates
3
-PO4 ends that would not be labeled by the conventional
TdT technique) in the fiber denucleation process in chicken
lenses has been invoked to explain this discrepancy (Torriglia et al., 1995
). The present experiments were also designed to resolve this issue, using a combination of enzymatic labeling and electrophoretic techniques.
Materials and Methods
). Fixed
lenses were routinely sectioned at 200 µm.
-dihexyloxacarbocyanine
iodide (DiOC6; Molecular Probes). SYTO 17 is a fluorescent DNA probe
with spectral properties similar to Texas red. DiOC6 is a lipophilic membrane probe with fluorescein-like fluorescence. We have used DiOC6 previously to visualize membrane systems in the embryonic lens (Bassnett,
1995
). After staining, the lens slices were washed in TBS for 30 min and
viewed with the confocal microscope using the 488-nm laser line and a
515-565-nm band-pass filter for DiOC6 fluorescence and the 568-nm laser
line and a 590-nm long pass filter for SYTO 17 fluorescence.
; Bassnett
and Shiels, 1996
). The nuclear lamina was visualized using a monoclonal
antibody raised against human lamin B2 (clone NCL-LAM-B2; Novocastra Laboratories Ltd., Newcastle upon Tyne, England). This antibody was
only effective on fixed lens slices following the high-temperature antigen
retrieval technique (Shi et al., 1991
). This involved immersing lens slices in
0.01 M citrate buffer, pH 6.0, and heating at full power for 10 min in a 600-W
domestic microwave oven. The lens slices were then allowed to cool, rinsed
briefly in PBS, and processed for immunofluorescence as described above.
-OH ends of DNA molecules by the enzyme
TdT. Preliminary experiments revealed significant nonspecific labeling of
nuclei in the absence of the TdT enzyme. It was necessary to include a
washing step (gentle agitation in 0.1% SDS at 60°C for 30 min) to remove
these unincorporated fluorescent nucleotides. The washing procedure reduced nonspecific labeling to low levels. In each experiment, some lens
slices were incubated in labeling mixture lacking TdT, as a negative control.
-OH termini.
Because there is evidence for DNase II-like activity in the chicken lens
(Torriglia et al., 1995
), we pretreated some slices with calf intestinal alkaline phosphatase (CIAP; GIBCO BRL, Gaithersburg, MD). CIAP treatment should convert any 3
-PO4 termini (produced by DNase II digestion)
to 3
-OH termini, the latter being detectable by the TdT assay. Permeabilized slices were incubated for 1 h at 50°C in 100 µl of dephosphorylation buffer (supplied with the enzyme) containing 1 µl (26 U) of CIAP. As a
positive control, some lens slices were first incubated for 30 min at 37°C
with 0.5 U/µl micrococcal nuclease (Boehringer Mannheim Corp.), an enzyme that produces DNA fragments with 3
-PO4 termini.
; Singh et al., 1988
), including lens epithelial cells (Kleiman and Spector, 1993
). Single-strand scissions are 5- to
2,000-fold more numerous than double-strand breaks in damaged DNA
(Bradley and Kohn, 1979
) and are readily detected under denaturing alkaline conditions. Lenses were fixed in 0.5% paraformaldehyde/PBS, pH
7.4, for 30 min at room temperature, embedded in 4% agar/PBS, and
sliced at 100 µm. Slices were reembedded in 0.3% agarose. The agarose
block was trimmed to a cube with a volume of <1 cm3. The agarose cube
containing the lens slice was immersed in permeabilizing buffer (1% sodium sarcosinate, 1% Triton X-100, 2.5 M NaCl, 100 mM EDTA, 10 mM
Tris, pH 10) for 1 h and then soaked in electrophoresis buffer (10 mM
EDTA, 0.4 M NaOH) for an additional hour. Some slices were incubated
in DNase I (50 U/ml) for 2 h at 37°C after the permeabilization step as a
positive control. The agarose cube was transferred to a horizontal gel electrophoresis apparatus and oriented so that the lens slice was perpendicular to the applied electric field. Electrophoresis was performed for 15 min
at 1.5 V/cm. After electrophoresis, the agarose cubes were neutralized in
0.5 M Tris, pH 7.5, for 30 min, stained with ethidium bromide (1 µg/ml)
for 45 min, and destained in distilled water. Blocks were transferred to the
stage of the confocal microscope and viewed using the 488-nm line of an
argon/krypton laser and a 515-nm long-pass dichroic filter.
Results
Fig. 2.
The fate of the nuclear membrane and DNA in fiber cells near the
border of the OFZ. The lens slice was
stained with DiOC6 (for membranes)
and SYTO 17 (for DNA) and viewed
with a confocal microscope. Cortical fiber cells are shown in A-C, cells just outside the border of the OFZ are shown in
D-F, cells immediately within the OFZ
are shown in G-I, and cells in the center
of the lens are shown in J-L. A, D, G,
and J are differential interference contrast images; B, E, H, and K, are confocal images of the DiOC6 fluorescence;
and C, F, I, and L are the corresponding
confocal images of the SYTO 17 fluorescence. Note the sudden change in nuclear morphology at the border of the
OFZ and the loss of the nuclear membrane (D-F). The last nucleus to possess
a nuclear membrane is indicated by the
arrow in D-F. Collapsed nuclei are evident in cells within the OFZ and SYTO
17-stained nuclear remnants (L, arrows)
can be discerned, even in the most central fiber cells. Bar, 25 µm.
[View Larger Version of this Image (113K GIF file)]
Fig. 3.
Confocal micrographs of lamin B2 immunofluorescence in an E17 lens slice after high-temperature antigen retrieval (see text for details). (A) Fiber cells in the peripheral cortex. The immunofluorescence is restricted to the lamina of the fiber cell nuclei. (B) Lowmagnification view of cells near the border of the OFZ. Note that even nuclear remnants (arrowhead) are positive for lamin B2 immunofluorescence. (C) High-magnification view of cells immediately adjacent to the OFZ (B, arrow). In many of these cells, the nuclear
lamina has become distorted, and the profiles of the nuclei are irregular (arrows), although some nuclei still retain a more normal appearance (arrowhead). (D) High-magnification view of cells immediately within the OFZ (B, arrowhead). At this point in the denucleation process, the fiber nuclei have collapsed into small spherical structures (arrows), and rents are apparent in the nuclear lamina. Bars: (A) 25 µm; (B) 50 µm; (C) 10 µm; (D) 5 µm.
[View Larger Version of this Image (88K GIF file)]
Fig. 4.
DIC and corresponding confocal immunofluorescence images of histone distribution in an E17 lens slice. The antibody recognizes an epitope common to histones H1, H2A, H2B, H3, and H4. (A) DIC image of the equatorial region of a lens slice showing the annular pad (ap) and superficial fibers. (B) Immunofluorescence image of the region shown in A. Note the strong labeling of nuclei in the annular pad and superficial fibers. (C) DIC image of fiber cells at the border of the OFZ. Fiber cell nuclei at various stages of disassembly are
present, including those with marginalized (arrow) or condensed chromatin (arrowhead). (D) Immunofluorescence image of the region
shown in C. During fiber cell denucleation, the distribution of histone proteins parallels that of the DNA (compare with Fig. 2 F ), becoming
first marginalized (arrow) and then collapsing into condensed residual structures (arrowhead). Bars, (A and B) 50 µm; (C and D) 10 µm.
[View Larger Version of this Image (117K GIF file)]
-OH ends of DNA
fragments. Labeled nuclei were rarely observed in the epithelial cell layer and never in the superficial fiber cells.
However, strongly labeled nuclei were always detected at
the border of the OFZ in lenses from embryos E15 and
older (Fig. 6 A). At earlier developmental stages, labeled
nuclei were not observed in the fiber cells. If the labeling
reaction was performed in the absence of the TdT enzyme,
no labeling was observed (data not shown). Surprisingly, only fully condensed nuclei were labeled. Irregularly
shaped nuclei with heterogeneous or marginalized chromatin were not labeled by this technique. End-labeled
DNA fragments were seen to persist in the cytoplasm of fiber cells hundreds of cell widths inside the border of the
OFZ. The lack of labeling in the more superficial fibers
could have been due to the presence of an inhibitor of the
TdT assay in these cells. To control for this, some slices were preincubated in DNase I to cause extensive DNA
fragmentation in vitro. In DNase I-treated tissue, all of
the nuclei were labeled by the TdT assay, including those
of the epithelial cells and annular pad (data not shown)
and superficial fiber cells (Fig. 6 B). The pattern of DNA
damage induced by the DNase I treatment was qualitatively different from that occurring naturally at the border
of the OFZ. In DNase I-treated cells, the labeling was heterogeneous and strongest immediately beneath the nuclear membrane (Fig. 6 B). In contrast, the collapsed nuclei of cells at the border of the OFZ were homogeneously
stained. The fact that all nuclei in the lens were labeled after the DNase I treatment suggests that the lack of label in
the outer cells of untreated lenses was not due to the presence of an inhibitor of the TdT assay in these cells.
Fig. 5.
Merged confocal image of fiber cells immediately
inside the border of the OFZ
after staining with DiOC6
(green) and SYTO 17 (red).
The combination of these fluorescent probes allows the simultaneous visualization of
the fiber cell membranes
(DiOC6) and nuclear DNA
(SYTO 17). At this point in fiber cell differentiation, the nuclei have collapsed into condensed spherical structures. Remnants of the nuclear membrane are still visible (arrowheads), attached to the naked
chromatin. The highly interdigitated lateral membranes
of the fiber cells are also evident. Bar, 5 µm.
[View Larger Version of this Image (155K GIF file)]
Fig. 6.
Merged confocal and DIC images of lens slices after TdT labeling with fluorescein-dUTP. The DIC images are shown in green and positively labeled nuclei (containing fragmented DNA) are shown in red. (A) At the border of the OFZ, the nuclei lose their regular shape (arrows) and collapse into condensed structures that are strongly labeled by the TdT assay (arrowheads). Positively labeled debris, resulting presumably from the disintegration of labeled nuclei, extends deep into the OFZ. (B) Cortical fiber cells from a lens slice
that was pretreated for 30 min with 50 U/ml DNase I. Note that after DNase I treatment, all nuclei are labeled by the TdT assay and that
the labeling is strongest immediately beneath the nuclear membrane. (C) Equatorial region of a lens slice that had been incubated with
CIAP before TdT labeling. None of the nuclei are labeled, indicating that the superficial fibers do not contain fragmented DNA with 3PO4 termini (see text for details). (D) Equatorial region of a lens slice that was treated sequentially with micrococcal nuclease and CIAP
before TdT labeling. All the nuclei are labeled, demonstrating the efficacy of the CIAP technique for detecting fragmented DNA with
3
-PO4 termini. Bars: (A) 50 µm; (B) 10 µm; (C) 50 µm; (D) 50 µm.
[View Larger Version of this Image (110K GIF file)]
;
Torriglia et al., 1995
). DNA damage caused by DNase II
would not be detected by a conventional TdT technique
because TdT only catalyzes the addition of labeled nucleotides to 3
-OH termini. DNase II-like enzymes, however,
would be expected to generate 3
-PO4 termini. We modified the TdT assay by incorporating a pretreatment with calf intestinal alkaline phosphatase (CIAP) to allow the
detection of 3
-PO4 termini. A CIAP-treated lens slice is
shown in Fig. 6 C. There were no labeled nuclei in the epithelium or superficial fiber cells of CIAP-treated lens
slices. Positively labeled nuclei were observed at the border of the OFZ in CIAP-treated slices, although no more
were observed than in untreated lens slices (data not
shown). To verify the efficacy of the CIAP treatment, some lens slices were first incubated in micrococcal nuclease to
induce the formation of DNA fragments with 3
-PO4 termini. In lens slices treated in this fashion, all the nuclei
were positively labeled by the TdT assay. This indicates
that lack of labeling in CIAP-treated lenses reflected the
absence of 3
-PO4 DNA termini in the nuclei of superficial
fibers and epithelial cells.
). At this stage of development, the OFZ contained many condensed nuclei,
but there was no evidence of TdT labeling. Only 3 d later,
at E15, did the condensed nuclei become labeled by the
TdT assay. Thus, the spatial gap observed at E19, between
the disappearance of PDI immunofluorescence and the
onset of DNA degradation (Fig. 7 A), paralleled the temporal gap observed at earlier stages.
Fig. 7.
Confocal images of lens slices
showing the distribution of ER (green)
and degraded DNA (red). (A) The ER
(visualized by immunofluorescence with
a protein disulfide isomerase antibody)
is abundant in the superficial layers of
the lens but completely absent from the well-defined central OFZ. The fiber cell
nuclear membranes are also labeled by
this antibody. Degraded DNA is localized in condensed nuclear remnants,
scattered throughout the cytoplasm of fiber cells in the OFZ. Note the gap of
~90 µm between the last fiber cell to
contain ER (arrow) and the first to contain degraded DNA (arrowhead). e, epithelium; ap, annular pad. (B) Apoptotic
cells are occasionally detected in the anterior epithelium of the E9 lens. At this
stage of development, the epithelial cells
(e) and all of the fiber cells ( f ) contain
abundant ER. Apoptotic nuclei (arrows)
are strongly labeled by the TdT assay,
and small apoptotic bodies are often observed in the adjacent tissue (arrowheads). Note that, in contrast to the central fiber cells shown in A, the apoptotic
nuclei of the epithelial cells are found in
cells in which the ER is still present.
Bars: (A) 100 µm; (B) 25 µm.
[View Larger Version of this Image (91K GIF file)]
Fig. 8.
Diagram showing
the principle of in situ electrophoresis. (A) A permeabilized, alkali-treated lens slice
is embedded in a block of
0.3% agarose and placed in a
weak electric field. Electrophoretically mobile, fragmented DNA is driven from
the slice into the gel. The
preparation is stained with
ethidium bromide and transferred to the stage of a laser
scanning confocal microscope (LSM). The focal
plane of the microscope is
positioned midway up the
slice at the level of the fiber
cell nuclei. A hypothetical
view down the microscope is
shown in B, the OFZ appearing as a dark region in a strip of brightly stained nuclei. Fragmented DNA is visualized as streams of positively stained material emanating from regions of the lens where the DNA was sufficiently degraded to be rendered electrophoretically
mobile.
[View Larger Version of this Image (29K GIF file)]
Fig. 9.
In situ electrophoresis of an E15 lens slice. (A) Low-magnification image of an ethidium-stained lens slice after electrophoresis. The slice is embedded in a block of 0.3% agarose. The dark space between the two bright arms (arrowheads) represents the OFZ.
The arms are formed by the ethidium-stained fiber cell nuclei and are continuous with the band of annular pad and epithelial nuclei seen
dimly in the background. The uneven bright strip extending across the image is a reflection from the edge of the agarose block. The arrow depicts the direction of the electrical field. (B) Intermediate-magnification view of a lens slice that was pretreated with DNase I before electrophoresis to cause fragmentation of fiber cell DNA. Note the diffuse clouds of ethidium-stained material emanating from all
of the fiber cell nuclei. (C) Untreated lens slice showing two streams of ethidium-stained material emanating from nuclei immediately
adjacent to the OFZ. (D) High-magnification image of cells at the border of the OFZ. Brightly stained individual nuclei are visible. A
stream of ethidium-stained material can be seen issuing from cells immediately adjacent to the OFZ. Bars: (A) 500 µm; (B) 250 µm; (C)
100 µm; (D) 25 µm.
[View Larger Version of this Image (81K GIF file)]
Discussion
) or the activation of lens endonucleases. However, recent studies have been unable to
reproduce the original observation that DNA fragments
with 3
-OH ends accumulate during fiber cell differentiation (Chaudun et al., 1994
). Furthermore, transcripts for
DNase I (the enzyme most likely to produce such 3
-OH
ends) have not been detected in the lens (Hess and
Fitzgerald, 1996
). This has led to the possibility that lens
nuclei may be degraded through the action of a DNase II-
like enzyme. A DNase II-like enzyme has been identified
in the lens on the basis of activity assays and immunological approaches (Torriglia et al., 1995
).
; Bassnett, 1995
). This led us to ask whether
nuclear disassembly is also a rapid event. We now report
that nuclear breakdown is indeed a rapid event that begins
shortly before cells reach the border of the OFZ. Over the
space of 20-30 cells and a time frame of a few days, nuclei
undergo a dramatic transformation, involving marginalization and subsequent collapse of the chromatin, loss of integrity of the nuclear lamina and nuclear envelope, and, finally, fragmentation of the DNA. Both the TdT labeling
and in situ electrophoresis data suggest that DNA damage
occurs only near the border of the OFZ, in cells that have
already lost their nuclear membranes. Thus, our results
contradict those of Modak and Bollum (1972), who reported
DNA damage throughout the lens, albeit with increasing
severity towards the center. The DNA damage that we detected was in the form of 3
-OH termini. These observations are, therefore, at odds with those of Chaudun et al.
(1994)
, who did not observe 3
-OH termini in any region
of the embryonic chicken lens using a nick translation
technique to detect fragmented DNA in situ. Interestingly,
Chaudun et al. (1994)
did observe DNA fragmentation in
the central fiber cells in tissue samples that were not fixed
immediately after dissection. This raises the concern that
the TdT-positive nuclei detected in our experiments could be a tissue preparation artifact due, perhaps, to postmortem activation of endogenous nucleases in the inner fiber
cells. However, in the present study, tissue samples were
always fixed immediately, the location of labeled cells was
reproducible (the border of the OFZ), and positively labeled cells were never observed in lenses younger than
E15. Thus, it seems unlikely that the TdT labeling of cells
at the border of the OFZ that we have observed represents an artifact of tissue preparation. It is possible that discrepancies in the TdT-labeling results reported by various groups simply reflect species variation or subtle methodological differences. As our present results demonstrate, to observe positively labeled nuclei, it is important
to examine cells in the appropriate region of the lens and
at the appropriate stage of development.
-PO4 termini accumulated during
lens cell differentiation, as would be the case if a DNase
II-like enzyme was activated (Liao, 1985
). We were unable to detect 3
-PO4 termini in lens fiber nuclei under assay conditions where 3
-PO4 termini induced by micrococcal nuclease treatment were readily observed. Thus, we
found no evidence for the activity of a DNase II-like enzyme in the fiber cell denucleation process. We cannot rule
out, however, the possibility that the positively labeled nuclei in cells at the border of the OFZ contained 3
-PO4 termini in addition to 3
-OH termini, as would be the case if
both DNase I- and II-like enzymes were activated simultaneously at the border of the OFZ.
-OH ends and, ultimately, the release of nucleosome-sized fragments of DNA into the cytoplasm (Appleby and Modak, 1977
). This kind of DNA fragmentation
is often found in cells undergoing apoptosis, and, indeed,
some authors have suggested that lens denucleation represents a form of apoptosis (Chaudun et al., 1994
). However, despite some superficial similarities, lens denucleation appears to be distinctly different from classical apoptosis. In
mature lens fibers, the other cytoplasmic organelles have
already disappeared 2-3 d before DNA fragmentation occurs. In contrast, apoptosis in many cell types (including
the lens epithelium; Fig. 7 B) is characterized by the presence of organelles in the cytoplasm of cells in which the
DNA has already been extensively degraded (Cossarizza et al., 1994
; Watt et al., 1994
; Weis et al., 1995). Furthermore, there is no evidence of membrane blebbing or the
formation of apoptotic bodies in lens fiber cells, although
fiber nuclei can sometimes have a lobulated appearance as
they collapse (Fig. 5). We have also shown that enhanced
binding of annexin V to the cell membranes (a marker for
apoptosis in many cell types) does not occur at the time of
fiber cell denucleation (data not shown). The time course
of lens denucleation (3-4 d from the onset of nuclear
changes to the complete disappearance of the DNA), although shown here to be more rapid than previously suspected, is nevertheless far slower than classical apoptosis.
Apoptosis in normal hepatic cells, for example, is completed in ~3 h (Bursch et al., 1990
) and overt DNA damage can be detected within 30 min of the application of the
apoptotic stimulus (Schmid et al., 1986
). Taken together, these data suggest that lens denucleation is morphologically and biochemically distinct from apoptosis. It is likely,
however, that these processes share fundamental regulatory mechanisms that determine whether the cells undergo
proliferation, apoptosis, or nuclear degradation. For example, expression of the viral oncoprotein, E7, in the developing lenses of transgenic mice causes inappropriate proliferation and triggers apoptosis in the fiber cells (Pan and Griep, 1994
). These effects can be inhibited by another oncoprotein, E6, possibly via its interaction with the tumor
suppressor protein, p53. Interestingly, nuclear degradation
is inhibited by expression of the E6 transgene, although by
a p53-independent mechanism (Pan and Griep, 1995
).
) alone is unlikely to account for the sudden degradation of DNA. Active degradation of DNA by fiber cell endonucleases remains the most plausible explanation for
fiber cell denucleation. A variety of candidate nuclease activities have been identified in embryonic chicken lens tissue. Muel et al. (1986)
have demonstrated the presence of
a Ca2+-dependent endonuclease in epithelial and fiber cell
nuclei. This activity may correspond to a DNase I-like,
30-kD protein identified in lens tissue by DNase activity
gels and immunoblots (Counis et al., 1991
; Arruti et al.,
1995
; Torriglia et al., 1995
). A 40-kD DNase, inhibited by
high concentrations of divalent ions but with an absolute
requirement for trace amounts of Ca2+ and Mg2+, has also
been demonstrated in both epithelial and fiber cells (Counis et al., 1991
) in addition to a cation-independent DNase active at neutral pH (Chaudun et al., 1994
). Three DNase I
isoforms have been identified in the lens by immunoblot.
All three isoforms (18-, 32-, and 60-kD bands) are present
in the epithelium, whereas the fiber cells contain the 32-kD
isoform only (Torriglia et al., 1995
). A DNase II-like activity has also been described in the embryonic lens (Torriglia et al., 1995
), where it is found in the cytoplasm of the
epithelial cells and the fiber cell nuclei and, surprisingly, in
the lens capsule, a cell-free extracellular matrix. This enzyme activity is evident only at low pH (5.5), but, as intracellular pH is known to decrease during fiber differentiation (Bassnett and Duncan, 1986
), a physiological role for
such an enzyme is not inconceivable. Immunoblot studies
have identified four different isoforms of DNase II in the
lens. The fiber cells contain bands of 18, 23, and 60 kD,
whereas two isoforms of 18 and 100 kD are found in the
epithelium (Torriglia et al., 1995
).
; Bassnett, 1995
), it is now possible to describe the events that lead to lens fiber denucleation and offer a hypothesis regarding the mechanism of
denucleation. It seems likely that a triggering event occurs
in the center of the lens on or about E12. The triggering
event could be related to the increasing size of the lens because the distance from the border of the OFZ to the lens
surface remains approximately constant through embryonic development. As the volume of the tissue increases,
diffusion-generated gradients of metabolites will be established within the lens as described by Bassnett et al.
(1987)
. A drop in cytoplasmic pH, a fall in oxygen tension,
or the build up of metabolic waste products are all candidates for the triggering event. We can speculate that the
trigger may act at the level of mitochondrial or ER membranes, as these appear to be lost first. The loss of these
major Ca2+ stores is expected to lead to an increase in cytosolic Ca2+ that may, in turn, mediate later events in the denucleation program. At this point, the nuclear lamina is
broken down, perhaps after phosphorylation of lamin protein substrates. The breakdown of the nuclear envelope
that occurs during denucleation may be similar to that
which occurs during mitosis, where the disassembly of the
nuclear lamina is known to result from the direct phosphorylation of lamins by the cdc2/cyclin B complex (Nigg, 1995
).
In this regard, it is interesting to note that Gao et al. (1995)
have demonstrated the presence of both cyclin B and the
cyclin-dependent kinase, p34cdc2, in E15 lens fiber cells.
After the breakdown of the nuclear envelope in the lens fiber cells, there is a delay before overt DNA breakdown
occurs. From a knowledge of the growth rate of the OFZ
(Bassnett and Beebe, 1992
), we can estimate that this delay is about 2 or 3 d. There are no obvious changes in nuclear
morphology during this period, but perhaps the structure of
the chromatin is undergoing transformation because, after
this time, extensive DNA damage is apparent. The fact that
DNA degradation was not observed until after the dissolution of the nuclear membrane raises the possibility that the
nucleus may be susceptible to invasion by cytoplasmic nucleases or factors that can activate previously quiescent
nuclear enzymes. Further experiments, where nuclei isolated from one region of the lens are mixed with cytoplasmic extracts from another, may help clarify this point.
Received for publication 12 August 1996 and in revised form 28 October 1996.
Address all correspondence to Steven Bassnett, Ph.D., Department of Ophthalmology and Visual Sciences, Washington University Medical School, 660 S. Euclid Avenue, Campus Box 8096, St. Louis, MO 63110-1093. Tel.: (314) 362-1604. Fax: (314) 747-1405. E-mail: Bassnetts{at}am.seer.wustl.eduWe thank Drs. Andley, Beebe, and Petrash for their comments on the manuscript.
These studies were supported in part by National Institutes of Health grants RO1 EY09852 and EY02687 (Core Grant for Vision Research) and an unrestricted grant to the Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc.
CIAP, calf intestinal alkaline phosphatase;
DIC, differential interference contrast;
DiOC6, 3,3-dihexyloxacarbocyanine iodide;
E, embryonic day;
OFZ, organelle-free zone;
PDI, protein disulfide isomerase;
TdT, terminal deoxynucleotidyl transferase.