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J Biol Chem, Vol. 274, Issue 27, 19338-19346, July 2, 1999
§,,
From the Department of Embryology, Carnegie
Institution of Washington, Baltimore, Maryland 21210 and
¶ Genetics Institute, Cambridge, Massachusetts 02140
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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We have developed a simple fluorescent assay for
detection of phospholipase A2 (PLA2)
activity in zebrafish embryos that utilizes a fluorescent
phosphatidylcholine substrate. By using this assay in conjunction with
selective PLA2 inhibitors and Western blot analysis, we
identified the principal activity in zebrafish embryogenesis as
characteristic of the Ca2+-dependent cytosolic
PLA2 (cPLA2) subtype. Embryonic
cPLA2 activity remained constant from the 1-cell stage
until the onset of somitogenesis, at which time it increased sharply.
This increase was preceded by the expression of a previously identified
zebrafish cPLA2 homologue (Nalefski, E., Sultzman, L.,
Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994)
J. Biol. Chem. 269, 18239-18249). By using a quenched
BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage
by PLA2, lipase activity was visualized in the cells of
living embryos where it localized to perinuclear membranes.
Phospholipase A2
(PLA2)1 catalyzes
the hydrolysis of the sn-2 fatty acyl bond of
glycerophospholipids to liberate lysophospholipid and free fatty acid,
both potential precursors to lipid signaling molecules (2-4). The
family of PLA2 enzymes is diverse and has been shown to
function in digestion of lipids, microbial degradation, inflammation,
cell signaling, and membrane remodeling (5). PLA2 enzymes
are primarily classified on the basis of their Ca2+
dependence and whether they are cytosolic or secreted (5). The
Ca2+-dependent high molecular mass (85 kDa)
cytosolic PLA2 (cPLA2) and some of the lower
molecular mass (28-40 kDa) Ca2+-independent cytosolic
PLA2s (iPLA2s) exhibit a substrate preference for phospholipids with arachidonic acid (AA) at the sn-2
position (6-8). This is of particular significance because AA released by PLA2 activity is the precursor for the synthesis of
eicosanoids, potent lipid mediators that include prostaglandins and
leukotrienes (2, 6, 9). In contrast, secreted PLA2s
(sPLA2s) and the 80-kDa cytosolic iPLA2 can
liberate AA, but they exhibit little preference for sn-2 AA
(5).
A number of studies have explored the contributions of different
PLA2 isoforms on total AA release and eicosanoid synthesis. In mice in which the cPLA2 gene was disrupted by targeted
mutagenesis, the production of prostaglandins, leukotrienes, and
platelet-activating factor were all reduced in peritoneal macrophages
(10, 11). Whereas the 80-kDa cytosolic iPLA2 can augment
cPLA2-induced AA release, work by Dennis and co-workers
(12, 13) suggests that its primary role is to remodel phospholipids.
Overexpression studies in tissue culture support the model that
following pro-inflammatory stimuli cPLA2 activation
triggers the immediate release of AA (within 30 min) that is essential
for a later, sPLA2-induced, phase of AA release (measured
over a 10-h period) (14-16). Whether or not sPLA2 plays a
central role in eicosanoid production, the above studies indicate that
cPLA2 is an important mediator of stimulus-induced AA
release and subsequent eicosanoid synthesis.
The cPLA2 protein is ubiquitously expressed in all adult
human tissues (17) but is subject to complex regulation that enables the immediate generation of eicosanoids in response to physiologic stimuli (18-20). For example, in cell culture, a number of receptor agonists (e.g. interleukin-1, ATP, and thrombin) increase
cytosolic calcium. Calcium promotes the rapid translocation of
cPLA2 from the cytosol to the membranes of the nuclear
envelope where it interacts with phospholipid substrates (1, 20-22).
Activity can be further enhanced by increased serine phosphorylation of cPLA2 by p42/p44 mitogen-activated protein kinases (18, 19, 23).
Although eicosanoids, the products of PLA2 activity, are
well known mediators of inflammatory and allergic responses in mammals (24) and teleosts (25, 26), their function during embryonic development
remains unclear. A developmental role is suggested by the presence of
cPLA2 activity in extracts of embryonic rat brain (27, 28)
and by the embryonic expression of cyclooxygenase, an enzyme that
utilizes the AA produced by PLA2 activity for prostaglandin synthesis (29, 30). Eicosanoids have also been implicated in
chondrogenic differentiation, bone metabolism, and palate development (31-34). However, targeted gene inactivation in mice has failed to
provide direct clues as to the developmental function of
cPLA2 activity. Mice deficient in a cPLA2
homologue do not have gross developmental abnormalities although they
are less likely to come to term (10, 11). The lack of a morphological
phenotype in these mice could be due to the expression and overlapping
function of two other recently identified cPLA2 homologues
(35).
As a first step in exploring the function of lipases in developing
embryos, we have devised an assay to measure PLA2 activity in embryonic extracts of the zebrafish, Danio rerio,
utilizing BODIPY-labeled phosphatidylcholine (PC) as a substrate. By
using this assay with different BODIPY-labeled substrates, and in the presence of various PLA2 inhibitors, we have identified
cPLA2 as the principal enzymatic activity in zebrafish
embryogenesis, and we characterized the developmental activity profile.
The finding that cPLA2 activity was the primary enzyme
responsible for embryonic activity was confirmed by Western analysis of
sucrose density gradient fractions. The zebrafish is an excellent model
system to study vertebrate embryonic PLA2 activity in
vivo in that embryos are optically clear and experimentally
accessible. These properties permit detection of enzymatic activity in
living embryos using a quenched BODIPY-labeled PC that fluoresces upon
cleavage by PLA2 (36, 37).
Materials--
1,2-Dipalmitoylphosphatidylserine (DPPS),
cholesterol, phosphatidylglycerol, phospholipase A2
(Naja mossambica mossambica), phospholipase C
(Bacillus cereus), and phospholipase D (type II, peanut)
were obtained from Sigma.
2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY C5-HPC; D-3803),
1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-2-hexadecanoyl-sn-glycero-3-phosphocholine (D-7707), and
1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY PC; B-7701) were obtained from Molecular Probes (Eugene, OR). Trifluoromethylarachidonyl ketone (AACOCF3),
methylarachidonyl fluorophosphonate (MAFP), and
(E)-6-(bromo-methylene)
tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol
lactone, BEL) were from Cayman Chemical Co. (Ann Arbor, MI).
Phenylbutylmethyl fluorophosphonate (PMFP) and trifluoromethylelaidoyl ketone (ECOCF3) were kindly provided by M. Gelb (University
of Washington, Seattle, WA).
Zebrafish--
Methods for breeding and raising zebrafish were
followed as described (38). Embryos were obtained from natural matings
of wild-type fish and staged according to criteria previously outlined (39).
Lipase Assay--
Single embryos were placed in 0.5 ml of embryo
medium (EM; in mM: 13.7 NaCl, 0.537 KCl, 0.025 Na2HPO4, 0.044 KH2PO4,
1.30 CaCl2, 1 MgSO4, 4.2 NaHCO3, pH
7.2 (38)), containing 150-200 ng of a fluorescent phospholipid
substrate, and sonicated for 2-5 s (6.3-cm cup horn, 100 watts; Heat
Systems-Ultrasonics Inc. Plainview, NY). After 2-4 h at 37 °C,
reactions were stopped by the addition of 1.5 ml of chloroform/methanol
(2:1) (40), mixed, and centrifuged (30 s, 16,000 × g).
The aqueous fraction was discarded, and the remaining lipid was dried
under vacuum, resuspended in chloroform, and loaded on thin layer
chromatography (TLC) plates (LK6D, Whatman, Clifton, NJ). Plates were
developed in toluene, diethyl ether, ethanol, and acetic acid
(50:40:2:0.2), and quantified using a laser scanner (blue laser, 800 V,
Storm 860, Molecular Dynamics, Sunnyvale, CA).
Yolk Removal--
Embryos (64-cell or shield stages) were placed
in Petri dishes containing EM and examined under a dissecting
stereomicroscope. The most posterior region of the yolk cell was
pierced with number 5 watchmaker's forceps. The wound was gently
reopened 1 or 2 times over the course of 10-20 min until most of the
yolk had flowed away from the blastoderm. Blastoderms were then rinsed
in EM and assayed as described. Zebrafish lipids were added to some
assays and were prepared as follows. Embryos (20 at shield stage) were homogenized in EM (50 µl) by sonication; methanol (100 µl) and chloroform (200 µl) were added, and the solution was mixed and centrifuged (30 s, 16,000 × g). The upper phase was
discarded, and the lower phase was dried under a stream of
N2. The lipids were resuspended in ethanol (2 µl)
followed by the immediate addition of EM (98 µl). Either one embryo
lipid equivalent (5 µl of lipid solution) or a control solution (5 µl of 2% ethanol in EM solution) was added to the assays.
Northern Analysis--
cPLA2 mRNA levels were
detected by Northern analysis (41) using a 32P-labeled
cPLA2 probe prepared by random priming (Prime-It II; Stratagene, La Jolla, CA) of gel purified (41) zebrafish
cPLA2 cDNA (2.5 kilobase pairs) excised from the PDE4
vector (Genetics Institute, Cambridge, MA).
Western Analysis--
Zebrafish embryos were homogenized in
buffer (50 mM Tris, pH 8.0, 80 mM NaCl, 1 mM EDTA, 1 mM EGTA), subjected to
SDS-polyacrylamide gel electrophoresis, and detected by Western
blotting using a polyclonal antiserum (1:1000) to human
cPLA2 (Genetics Institute, Cambridge, MA) and a goat
anti-rabbit secondary (1:2000, Amersham Pharmacia Biotech).
Immunoreactive bands were visualized by chemiluminescence (ECL+,
Amersham Pharmacia Biotech). In some experiments, embryos were
homogenized in buffer that also contained 5% sucrose and loaded on a
linear sucrose gradient (2 ml, 5-23%) for centrifugation (2 h at
259,000 × g). Fractions (130 µl) were collected, and
aliquots were assayed for total protein, PLA2 activity, and
by Western blotting as described above.
In Vivo PLA2 Assay--
Bis-BODIPY PC was purified
by TLC to remove low abundance BODIPY-labeled degradation products
immediately prior to incorporation into liposomes. The liposome
composition that yielded the best quenching was based on two previously
published protocols (36, 37). Briefly, a mixture of
DPPS/cholesterol/phosphatidylglycerol/bis-BODIPY PC (107:31:20:1; a
total of 287 nmol) was dried under N2 and resuspended in
ethanol (10 µl) followed by the addition of EM (10 µl). The solution was again dried under N2 until the volume was
approximately 7 µl to ensure the evaporation of most of the ethanol.
EM (0.15 ml) was added, and liposomes were prepared by sonication of
the mixture on ice (20 min, probe type). Liposomes were incubated with
dechorionated gastrulating embryos (2-4 h at 28 °C; 20 µl/embryo). Embryos were rinsed with EM and placed in a glass
depression slide. Fluorescence was visualized using a Leica TCSNT
confocal microscope.
Zebrafish Embryonic PLA2 Activity Is Detected Using a
Fluorescent Assay--
We have developed a sensitive assay for
PLA2 activity that utilizes a PC substrate containing a
BODIPY-labeled fatty acid on the sn-2 position. Cleavage of
this lipid by PLA2 liberates a fluorescent fatty acid that
is readily separated from the uncleaved substrate on TLC plates and
quantified by laser scanning. The addition of purified PLA2
(N. mossambica mossambica) to the assay buffer (EM + substrate) resulted in complete cleavage of the substrate, detected as
the appearance of a band on the TLC plate that co-migrates with free
BODIPY-labeled fatty acid (Fig. 1A,
lane 1), the expected product of PLA2 activity (Fig.
1A, lane 2). To test whether other phospholipases generate
products that also co-migrate with the free BODIPY-labeled fatty acid,
we examined the cleavage products of purified phospholipase C and
phospholipase D in the assay. Samples incubated with phospholipase C
produced a distinct fluorescent diacylglycerol species that was easily
resolved from the free BODIPY-labeled fatty acid (Fig. 1A, lane
3). The expected product of phospholipase D activity, a
fluorescent phosphatidic acid species, migrated with the uncleaved PC
substrate and not with the free fatty acid generated by
PLA2 (data not shown). In samples without added enzyme, we
observed a consistent small amount of nonenzymatic hydrolysis of the
substrate (0.03-0.05% of the substrate/h) which is 10-100-fold less
than that observed with samples containing PLA2 (Fig.
1A, lane 5). For this reason, all experiments included at
least one control incubation that consisted of assay buffer alone.
Following quantification of the TLC plate, the fluorescence of this
control reaction was used as the base line for all samples.
By using this assay, PLA2 activity could be detected in
extracts of single zebrafish embryos (Fig. 1A, lane 4).
Zebrafish PLA1 activity was not detected in these assays
using a TLC solvent system that resolves fluorescent lyso-PC (the
expected product of PLA1) from the fluorescent PC substrate
(data not shown). To test whether any PLA2 was inactivated
during the incubation period (typically 2-3 h), zebrafish embryonic
extracts were assayed for various lengths of time. The rate of
substrate cleavage was independent of assay duration for more than
7 h (Fig. 1B), indicating that the PLA2
activity in zebrafish embryonic extracts was stable.
PLA2 Activity Is Predominantly in the Cellular
Blastoderm of the Gastrulating Embryo--
Shortly after
fertilization, the zebrafish zygote segregates into two domains, the
dividing cells (blastodisc) atop a yolk cell (39). The blastodisc
divides multiple times generating a cellular blastoderm (approximately
4000 cells) that continues to divide while migrating over the large
(~700-µm diameter) yolk cell in a process termed epiboly (42).
Based on the yolk composition of other teleosts (43, 44), the zebrafish
yolk likely contains lipoproteins and lipids. The observed
PLA2 activity of the zebrafish embryo might serve the
following two functions: it could be associated with degradation of
yolk lipid for a nutritive purpose, or it could be a mediator of cell
signaling in the blastoderm. To distinguish between these
possibilities, the yolk was separated from the overlying blastoderm by
piercing the yolk cell and extruding the yolk (Fig. 2A). Levels of
PLA2 activity in the blastoderm and intact embryo were then
compared. Whereas we observed a small reduction in activity after yolk
removal in embryos assayed at the 64-128-cell stage (31 ± 5%),
no reduction was observed in shield stage embryos (6 h
post-fertilization, approximately 16,000 cells) (Fig. 2B).
Shield stage embryos and blastoderms were also assayed in the presence of additional zebrafish lipids to quantify the activity in the blastoderm and the levels of endogenous zebrafish lipid that can compete in the assay (Fig. 2B, and see "Appendix").
Modeling of these data indicate that the yolk contains no
PLA2 activity at shield stage (see "Appendix") and
suggests that embryonic activity primarily mediates cell signaling in
the blastoderm rather than yolk lipid processing.
cPLA2 Is the Principal Activity Detected in Zebrafish
Embryonic Extracts--
To characterize the phospholipase activity
present in zebrafish embryos, we utilized the fluorescent TLC assay to
analyze substrate preference, calcium dependence, and response to
specific inhibitors. Two substrates with different acyl chains were
useful to distinguish between activity due to sPLA2 or to
cPLA2/iPLA2 (D-7707 contains a 14-carbon
saturated fatty acid on the sn-2 position and a
BODIPY-labeled fatty acid on the sn-1 position, and D-3803
contains a BODIPY-labeled saturated fatty acid on the sn-2
position and an unlabeled fatty acid on the sn-1 position) (Fig. 3A). sPLA2
is known to exhibit little specificity for particular acyl chains, and
accordingly, purified snake enzyme cleaved both substrates in the assay
(Fig. 3B, lane 2 upper arrow and lane 5, lower
arrow). In contrast, purified human cPLA2 showed a
strong preference for D-3803 (Fig. 3B, lane 3) over D-7707
(Fig. 3B, lane 6). The BODIPY-labeled acyl chain of D-3803
was cleaved by cPLA2 30-fold more readily than was
saturated fatty acid of D-7707 (Fig. 3A, lane 3 versus lane 6).
When D-3803 was used a substrate, there was a linear relationship
between enzyme concentration and the level of PLA2 cleavage product; as little as 20 ng of human cPLA2 extract
generated significant BODIPY-labeled fatty acid (3-fold greater than
blank, Fig. 3C). This level of activity is in the range of
that previously observed for cleavage of phospholipids containing
radiolabeled AA (22) and confirms our observation that the affinity of
cPLA2 for, and cleavage of, D-3803 is comparable to that of
endogenous cPLA2 substrates. We hypothesize that the
hydrophobic BODIPY moiety at the sn-2 position disrupts the
local membrane environment in a fashion similar to AA.
The substrate preference of the enzymatic activity present in zebrafish
extracts (Fig. 3D) was more similar to that of the purified
human cPLA2 (D-3803 is cleaved to release a BODIPY-fatty acid, whereas D-7707 is not cleaved), suggesting that zebrafish embryos
do not contain significant sPLA2 activity. Consistent with
this observation, extract PLA2 activity was not
significantly inhibited by dithiothreitol (10 mM), a potent
sPLA2 inhibitor (treated embryos had 87 ± 9% of
control activity, n = 3, p > 0.4).
Since cPLA2 and some iPLA2s can exhibit similar
acyl chain preferences, comparisons of the cleavage rates of different
substrates is insufficient to discriminate between their activities.
One defining characteristic of iPLA2s is their
Ca2+ independence. The PLA2 activity in
zebrafish embryonic extracts was reduced by greater than 80% (Fig.
4A) with the removal of free
Ca2+ through the addition of EGTA (10 mM),
suggesting that the activity was predominantly due to cPLA2
and not iPLA2.
To confirm this observation, zebrafish extracts were incubated with a
panel of PLA2 inhibitors that are known to exhibit
different relative potencies for the two classes of enzyme. MAFP, an
irreversible inhibitor of cPLA2 that is believed to form a
covalent bond with serine 228 of the catalytic site (45), was a potent
inhibitor of the zebrafish extract activity (Fig. 4B).
Similarly, the well characterized competitive cPLA2
inhibitor AACOCF3 (46, 47) was also effective in blocking
extract activity (Fig. 4C). The substitution of the
arachidonyl moiety of MAFP with a phenyl-butyl group (PMFP) or the
arachidonyl moiety of AACOCF3 with an elaidoyl moiety
(ECOCF3) yields inhibitors that are more potent against iPLA2.2 However,
these modified inhibitors were less effective than their arachidonyl
versions at inhibiting extract activity (Fig. 4, B and
C). In sum, the ranked potency of PLA2
inhibitors for zebrafish extracts (MAFP (ED50 2 nM) > PMFP (ED50 200 nM) > AACOCF3 (ED50 15 µM) > ECOCF3 (ED50 30 µM)) more closely
paralleled that expected for cPLA2 than that expected for
iPLA2 (PMFP > MAFP > ECOCF3 > AACOCF3).
An additional inhibitor, BEL, is a potent and irreversible inhibitor of
iPLA2 that can also inhibit cPLA2, but only at
higher doses (14). In zebrafish extracts, BEL was 1000 times less
effective an inhibitor of PLA2 activity than MAFP.
Moreover, inhibition of the zebrafish activity required 50-fold higher
doses than reported for purified iPLA2 (48, 49) (Fig.
4D). Taken together, the pharmacological profile of the
PLA2 activity in the zebrafish embryo is most consistent
with the cPLA2 subtype.
Developmental Profile of PLA2 Activity and
cPLA2 Gene Expression--
PLA2 activity in
lysates was characterized during different stages of zebrafish
embryonic development. The activity observed at the 1-cell stage
reflects a significant maternal contribution of cPLA2
protein to the embryo (note that zygotic transcription does not begin
until the 1000-cell stage) (Fig.
5B). Activity remained
constant from the 1-cell to 4-somite stage and then increased, reaching
250% of initial levels in 24-h-old embryos. However, total embryonic
protein levels remained unchanged during this period (at the 1-cell
stage 27.1 ± 0.3 µg/embryo and after 24 h 24.2 ± 2.1 µg/embryo). PLA2 activity continued to climb to levels 8-fold greater than 1-cell stage embryos by 50 h
post-fertilization.
To correlate embryonic PLA2 activity with cPLA2
gene expression, we measured the RNA level of a zebrafish
cPLA2 homologue (1). To date, only this cPLA2
homologue has been described for zebrafish; however, additional closely
related genes are expected to be found (50). We examined expression of
this gene in relation to the enzymatic activity profile. A Northern
blot produced from RNA of staged zebrafish embryos revealed a
9.7-kilobase pair transcript that was weakly detected at the end of
gastrulation but rapidly increased to a constant level throughout
somitogenesis (Fig. 5A). Whereas the rise in zebrafish
extract PLA2 activity (between 12 and 20 h) was
preceded by the expression of this cPLA2 homologue (initially detected at 9 h), we cannot rule out that the products of other zebrafish cPLA2 homologues contribute to extract activity.
cPLA2 Immunoreactivity Co-migrates with
PLA2 Activity--
To confirm that the zebrafish activity
was due to cPLA2, embryonic extracts were subject to
SDS-polyacrylamide gel electrophoresis and Western blot analysis using
antiserum to human cPLA2 (Fig. 6). The cross-reactive antiserum detects
the predominant zebrafish product as a single 85-kDa band (lower
arrow, lane 2). We were unable to test the activity associated
with this band because cPLA2 activity from both zebrafish
extracts and purified human protein was poorly reconstituted from gel
or nitrocellulose fragments. However, extracts were subject to sucrose
gradient sedimentation, and fractions were assayed for PLA2
activity, total protein, and cPLA2 immunoreactive product.
Activity was observed primarily in fractions 2-4, whereas much of the
total protein was contained in fractions 3-5 (Fig. 6B).
More importantly, cPLA2 immunoreactive bands were only
detected in fractions that had high levels of PLA2 activity
(Fig. 6C).
PLA2 Activity Localizes to Intracellular Membranes in
Vivo--
We sought to develop an in vivo reporter of
PLA2 activity to visualize enzymatic activity in living
zebrafish embryos. Previously, a quenched BODIPY-labeled PC (Bis-BODIPY
FL C11-PC) had been used to visualize PLA activity in cultured
neutrophils (36). Bis-BODIPY FL C11-PC is a molecule similar to D-3803
with an additional BODIPY moiety added to the acyl chain on the
sn-1 position. When Bis-BODIPY FL C11-PC is incorporated
into cell membranes, the close proximity of the two BODIPY-labeled acyl
chains attenuates fluorescence due to energy transfer between the
fluorophores (36). Upon cleavage by PLA2, one of the
BODIPY-labeled acyl chains is released from the membrane, thereby
separating the two BODIPY moieties and increasing fluorescence. In
light of this work and our finding that BODIPY-labeled acyl chains are
cleaved by cPLA2, we tested whether quenched Bis-BODIPY FL
C11-PC could label living zebrafish embryos and act as an in vivo biosensor of PLA2 activity.
To incorporate the quenched fluorophore, live blastula stage embryos (4 h post-fertilization) were soaked in liposomes containing Bis-BODIPY FL
C11-PC. The addition of cholesterol to the liposomes was found to
quench the fluorophore more effectively, and the use of DPPS, as
opposed to PC, helped facilitate the movement of the fluorophore to the
inner leaflet of the plasma membrane (data not shown). Immediately
following liposome labeling, fluorescence could be detected under low
power using an epifluorescence stereomicroscope (Fig.
7).
To test whether the observed fluorescence truly reflected
PLA2 activity, embryos were labeled in the presence of the
cPLA2 inhibitor MAFP. The concentration of MAFP needed to
inhibit cPLA2 activity in an intact embryo was determined
by bathing embryos in various concentrations of MAFP for 2 h,
washing away the inhibitor, and lysing the embryos for the standard
PLA2 assay. When MAFP was added after an embryo had been
lysed and sonicated in assay buffer (0.2-0.5 ml), PLA2
activity was inhibited with an ED50 of 2 nM
(Fig. 4B). However, if an intact living embryo was soaked in
MAFP for 2 h, a concentration of 5 µM was required
to inhibit 81 ± 9% (n = 3) of the
PLA2 activity. Incubation of embryos with liposomes in the
presence of MAFP (5 µM) inhibited specific fluorescent labeling of embryonic cells visualized either at low power using an
epifluorescence stereomicroscope (n = 3, 3-5
embryos/group/experiment; Fig. 7A) or using a confocal
microscope (data not shown).
We ruled out that this inhibition was due to nonspecific effects of
MAFP treatment since incubation of liposomes containing D-3803 in the
presence of MAFP (5 µM) had no effect on the extensive fluorescence observed with this unquenched BODIPY PC (n = 2, 3-5 embryos/experiment; data not shown).
The subcellular localization of the fluorescence was examined using
confocal microscopy. PLA2-induced fluorescence was observed in perinuclear and nuclear membranes of embryonic cells, consistent with the localization of cPLA2 protein in activated tissue
culture cells (21) (Fig. 7B). In all experiments,
perinuclear and nuclear labeling was observed (n = 6, 3-9 embryos/experiment); however, not all cells showed equal levels of
PLA2 activity. This pattern of labeling was blocked by
MAFP, indicating that the perinuclear labeling observed in living
embryos is due to PLA2 activity.
PLA2 enzymes hydrolyze phospholipids, thereby
releasing molecules that can mediate a number of important cellular
processes, yet the regulation and function of PLA2 activity
during embryonic development remains unclear. We have devised a
sensitive PLA2 assay utilizing a BODIPY-labeled PC that
requires few experimental manipulations and yields reliable
quantitative data on the activity present in embryonic extracts. The
assay exploits new advances in laser scanning technology to quantify
fluorescent products on TLC plates and is sensitive enough to detect
PLA2 activity from single zebrafish embryos. This is the
first time PLA2 activity has been characterized in the
developing zebrafish, a powerful model system to study vertebrate
development by both visual and genetic means. The development of this
fluorescent PLA2 assay provides the basis of future studies
to understand the role of embryonic activity.
The assay we developed facilitated the characterization of the
PLA2 activity in zebrafish embryos and indicated that the
predominant activity is due to cPLA2. First, the TLC-based
assay was used to characterize the acyl chain specificity of zebrafish
PLA2 activity and to demonstrate that embryonic extracts
exhibited an acyl chain preference and a dithiothreitol insensitivity
incompatible with sPLA2 activity. By using two distinct
BODIPY-labeled PC substrates, we found that the PLA2
activity in zebrafish extracts has a 30-fold higher preference for
phospholipid with a BODIPY-labeled acyl chain on the sn-2
position over an sn-2 saturated fatty acid. Consistent with
this observation, Clark et al. (22) observed that
cPLA2 preferred arachidonyl phospholipids almost 20-fold over phospholipids containing other sn-2 fatty acids in
membranes from U937 cells (22). Thus, the acyl chain preference of
zebrafish extract activity is similar to that observed with purified
human cPLA2 and unlike that of sPLA2.
Since both cPLA2 and the 28-40-kDa iPLA2s
exhibit acyl chain preferences for AA, pharmacologic experiments were
performed to distinguish between the contributions of their activities
in zebrafish embryos. The observation that EGTA blocked more than 80%
of substrate cleavage suggested that the zebrafish embryonic activity
was predominantly due to cPLA2. To rule out potential nonspecific effects of EGTA, a panel of competitive and irreversible PLA2 inhibitors was tested. The activity profile in the
presence of these reagents confirmed that cPLA2 was the
major PLA2 activity in zebrafish embryonic extracts.
The TLC assay was also used to examine the compartmentalization of
PLA2 activity within the embryo. Experiments on deyolked embryos suggested that PLA2 activity localizes primarily to
the developing blastoderm after 6 h of development. The
observation that some activity is lost (<35%) when the yolk is
removed from younger embryos leaves open the possibility of an early
role for PLA2 activity in the yolk. The level of endogenous
PLA2 substrate (3 pmol/µg), determined by modeling the
effect of adding exogenous zebrafish lipids to the assay (see
"Appendix"), was consistent with the levels of arachidonyl-PC
(approximately 3-5 pmol/µg) observed in the embryos of other
teleosts (43, 44). These data are consistent with the endogenous
embryonic substrate being arachidonyl-PC, the ideal cPLA2 substrate.
A number of questions remain regarding the origin of enzymatic activity
(maternal versus zygotic) and the relative contributions of
different cPLA2 isoforms that comprise zebrafish embryonic PLA2 activity. Expression of a zebrafish cPLA2
homologue is detected approximately 6 h after the onset of zygotic
transcription and at least 2 h before total embryonic
PLA2 activity begins to increase. We also found that
cPLA2 activity sediments as a single peak by sucrose
gradient centrifugation, a peak that also contains the predominant
cPLA2 species detected by Western blot analysis. Absolute identification of the cPLA2 isoform(s) responsible for
embryonic activity awaits the cloning of other zebrafish
cPLA2 family members and the generation of isoform-specific
antibodies for immunodepletion experiments.
The substrate specificity, pharmacological profile, and the
immunoblotting experiments indicate that zebrafish embryo extracts contain at least one cPLA2 homologue and that this
homologue, possibly in conjunction with other cPLA2 family
members, is responsible for most of the embryonic activity.
The zebrafish is an ideal vertebrate model system to study biochemical
processes during development because its embryos are rapidly
developing, accessible, and optically clear. These properties facilitate the use of fluorescently quenched lipid probes that can
directly report PLA2 activity in living embryos. By having established that PC containing a BODIPY-labeled acyl chain on the
sn-2 position was a good substrate for cPLA2, we
utilized a double-labeled and fluorescently quenched BODIPY PC to
examine the subcellular localization of cPLA2 activity in
living embryos. Cleavage of this fluorophore by either PLA1
or PLA2 results in enhanced fluorescence (36, 37). In
zebrafish extracts, any increase in fluorescence was expected to
reflect cPLA2 activity. Upon incorporation of the lipid
fluorophore into cell membranes, PLA2 activity is expected
to result in two fluorescent species as follows: a diffusible free
fatty acid and a fluorescent lysolipid that is assumed to be less
mobile (and would probably be rapidly reacylated to form a fluorescent
PC).
Laser confocal microscopy revealed PLA2 activity in the
outermost embryonic cells, known as the enveloping layer. The absence of a signal from deeper cell layers probably does not imply low cPLA2 activity in these regions but rather reflects the
failure of the fluorophore to penetrate the embryo. In the enveloping layer, PLA2 activity was localized to regions surrounding
the nuclear envelope. This observation is consistent with reports by
Glover et al. (21), who used anti-cPLA2
antiserum followed by immunogold electron microscopy to localize
cPLA2 protein in stimulated tissue culture cells to the
perinuclear region.
The cPLA2 inhibitor MAFP had no effect on the incorporation
of an unquenched fluorescent substrate, yet it completely blocked the
perinuclear labeling observed using the quenched PLA2
activity reporter. These data strongly suggest that the observed
fluorescence truly reflects PLA2 activity.
Our current data provide evidence of significant PLA2
activity in the blastoderm of developing zebrafish embryos. This
activity increases during development and is almost entirely due to
cPLA2. These data also indicate that cPLA2
activity can be monitored using BODIPY-labeled acyl chains in both
in vitro and in vivo continuous lipase assays.
Recently, we have used these assays in a genetic screen for modifiers
of PLA2 activity and are now analyzing potential mutants.
We are also developing methods to label living embryos more
effectively, with the goal of examining PLA2 signaling in
real-time and correlating activity with specific developmental events.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Fig. 1.
PLA2 activity is detected in
zebrafish embryonic extracts. A, TLC plate of
fluorescent lipid fractions. Lane 1, 100 ng of purified
BODIPY-C5 standard. Control incubations (150 ng of fluorescent PC
(D-3803) in EM (50-µl final reaction volume)) contained purified
PLA2 (8 units, N. mossambica mossambica)
(lane 2) or phospholipase C (6 units, B. cereus)
(lane 3). A single gastrula stage embryo was lysed and
incubated as in lane 1 (lane 4). A "blank"
incubation lacked embryos or purified enzyme but consisted of substrate
in EM buffer alone (lane 5). A second blank had no embryo
and was not incubated (lane 6). The plate was developed with
toluene/diethyl ether/ethanol/acetic acid (50:40:2:0.2) and was
visualized as described under "Experimental Procedures."
B, PLA2 activity in lysed zebrafish embryonic
extracts was constant for over 7 h. Each reaction contained 2 gastrula stage embryo equivalents (50 µg of protein) incubated for
the indicated times. The buffer composition and TLC plate development
were as described in A. Data shown are the results of a
typical experiment.
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Fig. 2.
Zebrafish cPL A2 activity is
localized to the blastoderm. The yolk was removed from shield
stage zebrafish embryos, and resultant blastoderms were incubated in
200 µl of EM containing D-3803 (120 pmol/reaction; 1 h) and
assayed as described under "Experimental Procedures." A,
intact shield stage zebrafish embryo (left). Blastoderm with
yolk removed (right). B, PLA2
activity in the intact embryo compared with the yolkless blastoderm.
Prior to the start of the experiment, embryo lipids were extracted as
described under "Experimental Procedures" and added to some assays.
Data represent means ± S.E., n = 3.
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Fig. 3.
Zebrafish embryonic PLA2 activity
shows a preference for a BODIPY-labeled acyl chain over a saturated
acyl chain. A, BODIPY-labeled PC substrates.
Arrow indicates site of PLA2 cleavage.
B, TLC plate of the fluorescent phospholipid metabolites.
Either purified sPLA2 (0.8 units, N. mossambica mossambica) or recombinant human
cPLA2 protein (500 ng + 0.2 embryo equivalents of extracted
embryo lipids) was incubated (100 µl of EM) with fluorescent PC
labeled on the sn-2 (200 ng; D-3803) or the sn-1
(200 ng; D-7707) position. The TLC plate was developed with
chloroform/methanol/acetic acid/water (50:30:8:4). The free fatty acids
ran above the substrate (left arrow), whereas the lysolipids
ran below the substrate (right arrow). C,
BODIPY-labeled sn-2 fatty acid is a good substrate for human
cPLA2. Various amounts of cPLA2 were added to
embryo medium (20 µl final reaction volume) containing D-3803 (15 µM) and incubated for 2 h. D, the
zebrafish enzymatic activity exhibits a substrate preference similar to
human cPLA2. A gastrula stage embryo was incubated (100 µl of EM) with D-3803 (200 ng) or D-7707 (200 ng).
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Fig. 4.
Embryonic extracts contain predominantly
Ca2+-dependent cytosolic PLA2
activity. Zebrafish embryonic extract (1 gastrula embryo
equivalent) was added to embryo medium (500 µl final reaction volume)
containing D-3803 (200 ng/reaction), in the presence of pharmacologic
agents, and was incubated for 4 h. Data represent means ± S.E. from 3 to 6 experiments. Error bars are shown when
larger than symbol. A, EGTA inhibits extract
PLA2 activity. The irreversible inhibitors MAFP and PMFP
(B) and the competitive inhibitors AACOCF3 and
ECOCF3 (C) are potent inhibitors of zebrafish
extract PLA2 activity. D, BEL is 1000-fold less
effective than MAFP.
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Fig. 5.
Increase in PLA2 activity during
somitogenesis is preceded by the expression of a
cPLA2 homologue. A, Northern blot of total
RNA (10 µg) from staged zebrafish embryos. Probe was an antisense RNA
transcribed from the entire ORF of the zebrafish cPLA2
cDNA. B, total PLA2 activity during
embryonic development. A single stage embryo was added to embryo medium
(500 µl final reaction volume) containing D-3803 (200 ng/reaction)
and incubated for 4 h. Developmental stages are shown for
reference below the developmental time. All stages shown are at the
same magnification (the 1-cell stage embryo is 800 µm in diameter).
Data represent means ± S.E. from 3 to 6 experiments. Error
bars are shown when larger than symbol.
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Fig. 6.
cPLA2 immunoreactivity is
detected in fractions of embryonic zebrafish. A, 20 ng
of purified human cPLA2 (lane 1) or 5 µg
(approximately 1/4 of a 24 h embryo) of zebrafish extract
(lane 2) was subject to SDS-polyacrylamide gel
electrophoresis and detected by Western blotting using antiserum to
human cPLA2. The sizes of molecular mass standards are
given in kilodaltons. B, zebrafish extract (200-300 µg)
was loaded on a linear sucrose gradient as described under
"Experimental Procedures." Fractions were collected, and aliquots
were assayed for total protein and PLA2 activity. Data
represent a means ± S.E. of 3 experiments. C, 35 µl
of each fraction were mixed with sample buffer, boiled, and assayed for
cPLA2 immunoreactivity as described above. Data shown are
from a typical experiment. Human cPLA2 protein
(std; 2 ng) was loaded in the far right
lane.
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Fig. 7.
Localized cPLA2 activity is
detected in enveloping layer cells in vivo. PLA2
activity was observed in zebrafish gastrulas that had been incubated
with liposomes containing 1,2-bis-BODIPY-phosphatidylcholine for 2 h, as described under "Experimental Procedures." Embryos were
visualized at the end of gastrulation (100% epiboly) using a
stereomicroscope equipped with epifluorescence. Embryos exhibited
fluorescence immediately after labeling. A, addition of MAFP
(5 µM) to the liposomes completely inhibited cellular
fluorescence. B, subcellular localization of fluorescence
was visualized using laser confocal microscopy. PLA2
activity was observed in the cells of the enveloping layer,
predominantly in the perinuclear membranes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Gelb for kindly providing ECOCF3 and PMFP and Drs. C. Felder, I. Johnson, R. Pagano, and Y. Zheng for expert advice. We also thank S. Fisher, J. Liang, O. Martin, S. Nowak, A. Rubinstein, and C. Wiese for critical reading of the manuscript and helpful suggestions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Postdoctoral Fellowship F32 NS10326 (to S. A. F.).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.
§ To whom correspondence should be addressed. Tel.: 410-554-1229; Fax: 410-263-6311; E-mail:Farber{at}mail1.ciwemb.edu.
2 M. Gelb, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PLA2, phospholipase A2; cytosolic PLA2; sPLA2, secreted PLA2; iPLA2, Ca2+-independent cytosolic PLA2; BODIPY (D-3803), 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; AA, arachidonic acid; D-7707, 1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-2-hexadecanoyl-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; AACOCF3, trifluoromethylarachidonyl ketone; MAFP, methylarachidonyl fluorophosphonate; PMFP, phenylbutylmethyl fluorophosphonate; ECOCF3, trifluoromethylelaidoyl ketone; DPPS, 1,2-dipalmitoylphosphatidylserine; BEL, bromoenol lactone; EM, embryo medium.
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APPENDIX |
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To estimate the PLA2 activity in the yolk and blastoderm, assays were performed with and without the addition of one embryo's equivalent of zebrafish lipid (see "Experimental Procedures"). The amount of BODIPY-PC cleaved by an extract containing an intact embryo was determined experimentally to be 12 ± 2 pmol, a blastoderm was 16 ± 2 pmol, an embryo plus additional embryo lipid was 8.7 ± 0.5 pmol, and a blastoderm plus additional lipid was 10.7 ± 0.1 pmol (Fig. 2B). These data were used to determine the amount of PLA2 activity in the yolk and the blastoderm, and the levels of endogenous lipids that can compete for the fluorescent substrate by solving the Equations 1-4,
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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Solution of Equations 1-4 yielded the following: Y, 
0.6
pmol/h/embryo; B, 20 pmol/h/embryo; LB,
30 pmol; and LY, 44 pmol.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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