High Calcium Zones at the Poles of Developing Medaka Eggs

8 Pages·1992·3.2 MB·English

by  R A Fluck

Reference: Biol. Bull. 183: 70-77. (August, 1992)
High Calcium Zones at the Poles
Medaka
of Developing Eggs
R. A. FLUCK2 A. L. MILLER1 AND L. F. JAFFE'
, ,
1Marine Biological Laboratory, Woods Hole, Massachusetts 02543 and'Department ofBiology,
Franklin & Marshall College. Lancaster, Pennsylvania 17604
Abstract. We have injected medaka fish zygotes with m-RNAs all attach to the vegetal pole ofthe very young
recombinant aequorin and visualized the resulting pat- oocyte; in which the zygote'sanimal halfisstill totipotent;
terns of luminescence to reveal patterns of free calcium and in which early blastula cells derived from the vegetal
during early development. We have co-injected fluores- pole plasm induce formation ofboth the embryonic me-
cein-labeled aequorin to correct for nonuniformities in soderm and the dorsal axis (Capco and Jeffery, 1982;
aequorin (as opposed to calcium) distributions by visu- Nieuwkoop, 1985; Wylie et ai, 1985; Forristall et al,
alizing the resulting patterns of fluorescence as opposed 1991; Melton, 1991). Yet another example is the Dm-
toluminescence. We havealsocoinjected acalcium buffer sophilaegg. in which early pattern isorganized from both
to facilitate calcium diffusion, dissipate apparent calcium poles and in which certain key m-RNAs, as well as the
gradients, and thus confirm their reality. germ plasm, movewithin thematuringoocytetotheinsect
An exploratory study shows zones ofelevated free cal- equivalent of the vegetal pole (Niisslein-Volhard and
cium at the vegetal as well as the animal pole during the Roth, 1989; Ephrussi et at.. 1991).
first day ofdevelopment and thus up to the beginning of Inall threeoftheseeggs ascidian, frog, and fly early
gastrulation. A closer study during the first 6 h, and thus patterning involvesastriking movement ofkey materials
through ooplasmic segregation and early cleavage, shows to, or near, the vegetal pole. In these animal eggs, more
a steady zone of high calcium at the vegetal pole and a is known about these key materials than about the mech-
slowly oscillating one at the animal pole. The latter is anisms that establish this pole and drive them to it; the
particularly strong during ooplasmic segregation and cy- reverse is true in the fucoid egg. Little is known of the
tokinesis. This report contains the first unambiguous ev- key materials in this plant egg; however there is strong
idenceofrelativelysteadyzonesofhigh cytosoliccalcium evidence that the establishment of a steady subsurface
during the development ofan animal egg. zone of elevated calcium is essential to the mechanism
that establishes the first in this case rhizoidal devel-
Introduction opmental pole. Moreover, such a zone has been directly
visualized at a stage when localization of the rhizoidal
thePavtetgeertnalfpoorlmeatioonf asntairmtaslategtghse. Oponleesstrpiakritnigcuelxaarmlyplaet polTehiesrstoillel orefvheirgshibcleal(cJiafufme,zo1n9e9s0ai,nbt)h.e polarizing fucoid
of this general statement is in the ascidian egg. Here the egg suggests that they may play a comparable one in an-
unfertilizedeggis ,ofreeofpattern thatanyhalfwill form imal eggs. Indeed, there is interesting evidence for just
a tadpole; yet after >'i<!ization and the resultant con- such a role in.Vtwj/>;oocytes(Robinson, 1979; Larabell
traction of the cortex M! a region near the vegetal andCapco, 1988)aswell asin Dwsophilufollicles(Overall
pole, onlyvegetal fiat will formtadpoles(Reverberi, and Jaffe, 1985) and in ascidian zygotes (Jeffery. 1982).
1971; Jeffery, 1984; S| ijder et at., 1990a). Another Moreover, in aequorin-loaded medaka eggs, persistent
example is the frog, in uli; pattern starts as the mito- polar zones of high luminescence were visualized more
chondrial cloud, thegerm plasm, and oneormore potent than six years ago. However their meaning has been un-
certain because ofthe possibility that the aequorin rather
Received4 February 1992;accepted 18 May 1992. than free calcium was concentrated at these eggs' poles
70
HIGH CALCIUM ZONES IN MEDAK.A EGGS 71
45 MIRROR MIRROR SUPPORT
MEDAKA
EGG '
/
COLOR SCALE
MM
1-2 photons
MM
MM 3-4 photons
5-6 photons
7-8 photons
Mo4iMi? 9-10 photons
11-25 photons
500pm
72
HIGH CALCIUM ZONES IN MEDAKA EGGS 73
tons per pixel with a color scale. Photon collection was the natural aequorin used in the older studies somehow
briefly(10s)interruptedatappropriateintervalstorecord inhibiteddevelopment beyond theblastulastage(Ridgway
brightfield imagesoftheeggs. Thisallowed ustocompare d al., 1977).
luminescent and brightfield images as the relatively slow Color Figure 4 displays this pattern during the first 2
processes ofooplasmic segregation and cytokinesis pro- h, almost to first cleavage; while Figure 7 provides quan-
ceeded. titative information as well as temporal detail from fer-
We used the IPD system to observe the fluorescence tilization through the first few cleavages. A strong vegetal
emitted by fluorescein-labeled aequorin in the eggs. Ap- zone ofluminescence appears as soon as the fertilization
propriateexcitation filters(two485 DF22 filters in series) wave reaches the vegetal pole and remains relatively con-
and a 530-DF30 barrier filterwere obtained from Omega stant in both intensity and diameter forat least 6 h. How-
Optical, Inc. (Brattleboro, Vermont). The latterwas sup- ever, a distinct animal zone of luminescence does not
OG
plemented with two Schott 530 filters. appearuntil well afterthe fertilization wave hassubsided.
Our quantitative analysis consisted of measuring lu- Itthen slowlyoscillatessoastolargelydisappearandthen
minescence in 150jum wide square boxes placed ateither reappearthree times during the first 6 h. These reappear-
the vegetal oranimal polesandthen dividingthese values ances presumably represent the slow calcium waves that
by the mean of those collected from two similar boxes accompany cytokinesis during the first three cleavages
placed at the equator ofthe egg (Fig. 2). Each resulting (Fluck et al., 1991).
luminescence ratio was divided by a corresponding flu- However, the biological role of the first, precleavage
orescence ratio (from fluorescein aequorin) to correct for zone ofanimal pole calcium is less clear. The additional
differences in aequorin concentration. All raw light in- examples displayed in Figure 6 indicate that this pre-
tensity values were corrected for background. cleavage zone is a real one that is present between about
20% and 50% ofthe period between fertilization and first
Results and Discussion cleavage. The presence ofthisprecleavage zoneofanimal
Color Figure 3 displays the pattern ofluminescence in pole calcium may be somehow connected to the process
a representative aequorin injected medaka eggduringthe of ooplasmic segregation because this process is largely
first 22 h ofdevelopment, up to the early to mid gastrula completed while this zone is present. Ooplasmic segre-
stage. Striking zones ofhigh luminescence persist at both gation comprises the roughly simultaneous movement of
the oil droplets toward the vegetal pole and ofthe bulk
poles throughout this period.
The blastodisc seems to roughly correspond to the an- of the cytoplasm to the animal pole. Both oil droplet
imal zone of high luminescence. However, no known movement and the thickening of the blastodisc, which
structure corresponds to the remarkable. 150 to 300 nm indicates cytoplasmic flow, start at about 20% ofthe pre-
wide vegetal zone ofluminescence. Moreover, we do not cleavage period and are largely over by 70% ofit (Fluck
yet know ifthe gradual, overall decline in luminescence et al.. unpublished).
during the first day ofdevelopment represents a slow fall
in aequorin or in free calcium concentration. However, Polar luminescence representsfoci ofcalcium
the observed pattern of luminescence should somehow not aequorin
represent a natural, developmental pattern because me-
daka eggs, which are loaded with recombinant aequorin, Color Figure 5 displays representative patterns offlu-
regularly hatch into swimming fish. While roughly con- orescence coming from eggs injected with fluorescein-ae-
sistent with the preliminary results reported earlier(Jaffe, quorinduringtheprecleavageperiod. Nosign ofavegetal
1986). these new ones should be more reliable because focus ofaequorin fluorescence can be seen.
Figure3. Representativepatternsofluminescencefromanaequorin-loaded medakaeggduringthefirst
dayofdevelopment. Eachcoloredpanelshowstheluminescenceaccumulatedduringtwosuccessivehours.
Thuspanel 3A showsluminescence accumulated for 2 h startingat 5 min after fertilization; 3B showsthe
luminescence accumulated from 2 to 4 h; 3C, from 4 to 6 h, etc. Panels 3A'. F'. and K'show sketchesof
theslowlyrotatingeggmadefrombrieftransmittedlightobservationscarriedoutjustafterthecorresponding
luminescentimageswereobtained. Thepersistentantipodal zonesofhigh luminescenceremain attheegg's
poles.
Theluminescenceseeninthelefthandhemisphereof3Arepresentsafaintresidueoftheintensefertilization
wavedescribedbyGilkeyelal.(\978)andbyYoshimotoelal.(1986). Itisscarcelyseenontherightbecause
the aequorin had not yet fully diffused to the right halfat this time. The interpolar spot ofdecreasing
luminescence in the left halfof panels 3A-C represents calcium leaking in through the slowly healing
injection site. It iswound calcium.
74 R. A. FLUCK. ET AL.
COLOR SCALE
1 photon 6 photons
2-3 photons 7-8 photons
4-5 photons 9-25 photons
Figure4. Patternsofluminescenceduringthefirst 2 hofdevelopmentofthesameeggshown in Figure
3. Each colored panel represents luminescence accumulated over successive 30-min periods: while each
black and white image was obtained via brieftransmitted light exposure soon afterthe corresponding lu-
minescentone. Thegrowingblastodisc's imageshave been tinted yellow to makethem easiertosee.
Figure5. Patternsoffluorescencefromarepresentative medakaeggcoinjected with bothaequonnand
fluorescein-labeled aequorin so as to indicate the distribution ofaequorin. A. Observed at 30 min after
fertilization, astage right afteraccumulation ofthe imageshown in Figure4A. Notetheabsence offoci of
fluorescenceateitherpole. The faint,apparent fluorescenceseenoutsideoftheegg'sboundary isanartifact
resulting from incomplete absorption ofthe exciting light by the barrier filter. (B) Observed 90 min after
fertilization, astageafteraccumulation ofthe imageshown in Figure4C. A focusoffluorescence has now-
appeared at theanimal pole because ofthe accumulation ofcytoplasm in the blastodisc duringooplasmic
segregation.
In uninjected controleggs, autofluorescence isso low equatorial or background luminescence when the lumi-
that the egg appears as a black hole against the dim nescence ratio is divided by the corresponding ratio of
extracellular glow produced by that exciting light that fluorescence coming from coinjected fluorescein-aequorin
traverses the barrier filter. So a vegetal focus of fluo- in the two regions. This inference was further confirmed
rescence from aequorin is not hidden by autofluores- by the results ofexperiments in which the eggs were in-
cence. Aequorin is simply not concentrated at the veg- jected with enough of the calcium buffer, dibromo-
etal pole. BAPTA to substantially facilitate the diffusion of Ca2+
Hence the vegetal zone ofluminescence must represent away from any zones of high [Ca:*]. which are in the
a standing zone ofhigh free calcium, not aequorin. The micromolar range. As Table II shows, such injections
data in Table I confirm thisinferencequantitatively. Veg- halve the relative intensity of the vegetal zone of lumi-
etal luminescence remainsthree times more intense than nescence.
HIGH CALCIUM ZONES IN MEDAKA EGGS 75
300 pole when averaged over time. This raises the question
ofhowgenerally high calcium zonesappearatthe vegetal
C poles ofdeveloping oocytes and eggs.
I It also raises a number ofother questions. Thus what
200- are the sources (and sinks) which maintain the high cal-
cium zones particularly the vegetal one in the medaka
0Q). egg? To what extent does this calcium come from the
medium, from the yolk compartment or from the endo-
plasmic reticulum?A relatedquestion concernsthe radial
o
location ofthese zones. Are they restricted to a very shal-
O.
low regionjust underthe plasma membraneasthey seem
tobe in fucoid eggs(Jaffe, 1990a)and in medakacleavage
furrows (Fluck el al.. 1991 )? Above all, what is their role
100 200 300 400
Minutes after Fertilization
Kigure6. Luminescent intensitiesobservedatthevegetal(O).animal
().andequatorial(+)regionsatvarioustimesafterfertilization. While 30
the focusofluminescence at the vegetal pole isclearly due to a steady
zoneofhigh calcium there, the focusat theanimal pole isonly clearly
above that due to more cytoplasm and hence more aequorin there at
theprecleavagepeak(a)andduringeachofthefirstthreecleavages(b- ,X
d).
o
Color Figure 5 alsodisplaysthe pattern offluorescence .QC.
coming from fluorescein-aequorin in the animal hemi- 20 40 60 80 100
sphere. Here an obvious focus offluorescence appears at Minutes after Fertilization
the animal pole together with the precleavage zone oflu-
minescence. Sothedevelopment ofa precleavage zoneof B
high animal calcium is not qualitatively obvious. How-
ever. Table I shows that when the ratio ofanimal pole to
equatorial luminescence iscorrected forthecorresponding
fluorescence ratio, it remains about five-fold and thus
clearly represents a second zone ofhigh free calcium at
theanimal pole. Moreover, asTable II and Figure8 show,
the reality ofthis second, animal zone is also confirmed
by the results of coinjecting a calcium buffer. When
enough is introduced to substantially facilitate the diffu-
sionofCa2+ away from anyzoneinthe micromolar range. Mi2n0utes 4a0lter F6e0rtilizat8i0on 100
the luminescence at the animal pole falls steadily to a
level which is far below the control level.
20
Significance ofthe results
This report shows that the zones ofhigh luminescence
seen at the poles of aequorin-injected medaka eggs are
indeed zones ofhigh free calcium. A preliminary report
is available ofa similar, ifless continuous, high calcium
zone at the vegetal pole ofXenopm eggs (Miller el al.,
1991). Moreover, in ascidian zygotes. striking, periodic
calcium waves are regularly seen to start at or near the 20 40 60 80 100
vegetal pole during the period between fertilization and Minutes alter Fertilization
first polarbody formation (Speksnijder el al.. I990b). Be-
cause these waves are attenuated en route to the animal Figure 7. Luminescent intensity observedat theanimal pole \-cr\it\
timeafterfertilizationinthreeeggs.(A)Thefirst 100minofdatashown
pole, theycorrespond toa high calcium zoneat the vegetal in Figure 7. (B, C) Data from two othereggs.
76 R. A. FLUCK ET AL.
Relativeluniiiiest.v. ',a theequatorofaequorin-loaded
medakaeggsinthe ' <'''
Luminescence ratio
SEM
pole/equator
Vegetal Animal
Uncon, 11 3.0 0.7 8.7 3.4
Corrected forAequorin Ratio" 9 3.4 1.4 4.9 1.6
3Luminescencewasaccumulated betweenabout 15 and 100mmafter
fertilization at 16Candthusbetween 10and60% oftheperiod before
cleavage.
bThiswasdoneinaseparategroupofeggscoinjectedwithfluorescein-
labeledaequonn bydividingtheluminescenceratiobythe fluorescence
ratio.
in development? In particular, what is their role in oo-
plasmic segregation? Heresomeevidence isalready avail-
able. Longago. Yoshi Sakai reported somestrikingeffects
of prick activation on the loci ofooplasmic segregation
in medaka eggs (Sakai. 1964). In particular, if vegetal
halves are strongly prick-activated, then the cytoplasm
accumulates (to form what would normally be the blas-
todisc) at the point ofactivation and the oil droplets mi-
grate towards the opposite pole. Such pricking undoubt-
edly both initiated an activation wave ofcalcium and es-
tablished an enduring region ofhigh calcium atthewound
(compare Fig. 3 and Fig. 4). Itistruethattheorganization
ofanimal halvesprovedtobelesssensitivetothepricking
site. Nevertheless, these old experiments suggest that cal-
cium zones may play at least a reinforcing role in normal
ooplasmic segregation. Recently, we have observed that
ooplasmic segregation in the medaka eggcan be markedly
inhibited by injecting enough ofthe calcium buffer, di-
bromo-BAPTA. to inhibit the formation ofhigh calcium
zones (Miller el a/., unpub.).
*
Table II
Dibromo-BAPTA reducesaequonn luminescenceat theanimal
ami vegetalpoles ofthemedaka egg3
Luminescence ratios
SEM
pole/equator
Injectate(s) Vegetal Animal
Aequonn
HIGH CALCIUM ZONES IN MEDAK.A EGGS 77
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