Gravity influences the position of the dorsoventral axis in medaka fish embryos (Oryzias latipes).

To determine whether gravity influences the plane of bilateral symmetry in medaka embryos, zygotes were placed with their animal-vegetal axis orientated vertically and with their vegetal pole elevated. Then, at regular intervals during the first cell cycle, the zygotes were tilted 90 degrees for about 10 min and subsequently returned to their original orientation. In embryos tilted during the first half of the first cell cycle, the embryonic shield formed on the side that had been lowermost when the zygote was tilted. In embryos that were tilted twice, first in one direction and then in the opposite direction, the embryonic shield formed on the side that was lowermost the first time. When zygotes were centrifuged at 5 g, the embryonic shield formed on the outwardly radial (centrifugal) side of the embryo. The orientation of the array of parallel microtubules in the vegetal pole region was also influenced by tilting or centrifuging zygotes. No correlation was found between the positions of the polar body and the micropyle and the plane of bilateral symmetry. It was concluded that gravity influences both the plane of bilateral symmetry and the orientation of microtubules in the vegetal pole region of medaka embryos.

Responses of the Medaka Fish Egg (Oryzias latipes) to the Photolysis of Microinjected Nitrophenyl-EGTA, a Photolabile Calcium Chelator.

Photolabile calcium chelators (calcium cages) can be used to elevate cytosolic [Ca2+] at specific sites and times (1, 2, 3). They have been especially valuable in flash photolysis studies of muscle contraction (2) and secretion (4, 5). In the present report, I describe several responses of medaka eggs to thephotolysis of microinjected nitrophenyl-EGTA (NP-EGTA),a new calcium cage (6). When unfertilized eggs injected with NP-EGTA were irradiated with ultraviolet irradiation in a small region of the egg, the eggs were activated and ooplasm within the irradiated region contracted and accumulated there. Eggs into which NP-EGTA was injected could also be fertilized. Subsequent irradiation of such eggs, in addition to causing the contraction and accumulation of ooplasm, also caused a global contraction of dividing blastomeres and the contraction and blebbing of embryonic cells for up to 4 days after fertilization. Injection of NP-EGTA had no apparent effect on the maturation offertilized eggs, which developed normally and hatched.

Microtubule-Based Movements During Ooplasmic Segregation in the Medaka Fish Egg (Oryzias latipes).

We used time-lapse video microscopy to monitor the effects of cytochalasin D (CCD) and demecolcine on cytoplasmic streaming toward the animal pole of the medaka egg, the formation of the blastodisc at the animal pole, the movement of oil droplets in the cytoplasm toward the vegetal pole, and the saltatory movement of small cytoplasmic parcels toward the animal pole and vegetal pole. Cytochalasin D inhibited both cytoplasmic streaming toward the animal pole and the formation of the blastodisc, suggesting a role for microfilaments in these processes. However, CCD had no apparent effect on saltatory movement or on the movement of oil droplets toward the vegetal pole. Thus, the segregation of oil droplets toward the vegetal pole is not the result of the bulk movement of ooplasm toward the animal pole. In eggs treated with demecolcine, oil droplets did not move toward the vegetal pole but instead floated to the uppermost portion of the egg, and saltatory movement was absent, suggesting that microtubules are required for these movements. The effects of demecolcine on oil droplet movement and saltatory movement could be reversed by irradiating the eggs with UV light (360 nm). Using indirect immunofluorescence, we showed that irradiation of demecolcine-treated eggs with UV light regenerated microtubules within the irradiated region. The specificity of the mechanism responsible for the vegetal poleward movement of oil droplets was assessed by microinjecting droplets of five other fluids--mineral oil, silicone oil, vegetable oil, and two fluorinated aliphatic compounds--into the ooplasm. None of these fluids segregated with the endogenous oil droplets. These results suggest that a specific mechanism, probably involving microtubules, is responsible for the segregation of oil droplets to the vegetal pole.

Microtubule Arrays During Ooplasmic Segregation in the Medaka Fish Egg (Oryzias latipes).

We used indirect immunofluorescence to study microtubule arrays in the medaka egg between fertilization (normalized time, Tn, - 0) and the first cleavage (Tn = 1.0). Eggs were fixed at various times after fertilization and examined with conventional fluorescence microscopy, laser scanning confocal microscopy, and three-dimensional fluorescence microscopy. Soon after the eggs were fertilized (Tn = 0.02), we saw microtubules oriented perpendicular to the plane of the plasma membrane but none parallel to the plasma membrane. Later (Tn = 0.08), we saw an array of microtubules oriented more or less parallel to the plasma membrane but having no apparent preferred orientation with respect to the animal-vegetal axis of the egg. In the interpolar regions of the egg, this network increased in density by Tn = 0.24 and remained a constant feature of the ooplasm until the first cleavage. From Tn = 0.30 to 0.76 the polar regions of the egg contained dense arrays of organized microtubules. At the animal pole, microtubules radiated from a site near the pronuclei; while at the vegetal pole, an array of parallel microtubules was present. Injection of the weak (KD = 1.5 {mu}M) calcium buffer 5,5`-dibromo-BAPTA disrupted the radial pattern of microtubules near the animal pole but had no apparent effect on the parallel array of microtubules near the vegetal pole. Because this buffer has previously been shown to suppress a zone of elevated cytosolic calcium at the animal pole and to disrupt ooplasmic segregation in this egg, the results of the present study (1) are consistent with a model in which microtubules are required for ooplasmic segregation in the medaka egg, and (2) suggest that the normal function of a microtubule-organizing center at the animal pole of the egg requires a zone of elevated calcium.

Calcium Buffer Injections Inhibit Ooplasmic Segregation in Medaka Eggs.

Injection of the weak (KD = 1.5 {mu}M) calcium buffer 5,5'-dibromo-BAPTA into fertilized medaka eggs inhibited the formation of the blastodisc at the animal pole, the movement of oil droplets toward the vegetal pole, and cytokinesis. These inhibitory actions were dependent upon the concentration of the buffer but were independent of free [Ca2+] in the injectate. Because this buffer has previously been shown to substantially suppress zones of elevated calcium at the animal and vegetal poles of the medaka egg, the results of the present study suggest that these zones are necessary for normal segregation of the ooplasm and its inclusions in the medaka egg.

Calcium buffer injections inhibit cytokinesis in Xenopus eggs.

A slow cortical wave of high calcium accompanies the elongation of cleavage furrows in medaka fish eggs as well as in Xenopus eggs. We explored the role of such waves by injecting calcium buffers into Xenopus eggs at various times before and during first and second cleavage. Injection earlier than about 15 minutes before first cleavage normally starts delays it for hours. Injection between about 15 minutes and a few minutes before cleavage normally starts allows a (short) furrow to form on time but usually yields an eccentric one. This forms away from the injection side, often as far off-center as the egg's equator, and then regresses. Injection soon after it starts quickly arrests elongation of the furrow and eventually induces its regression; while injection a bit later likewise soon arrests elongation but allows delocalized furrow deepening to continue. The dependence of these inhibitory actions upon the dissociation constants and final cytosolic concentrations of the injected buffers indicates that they act as shuttle buffers to suppress needed zones of high calcium in the micromolar range. We conclude that the high calcium that is found within these furrows is needed to induce them, to extend them and even to maintain them. Moreover, while short, eccentric furrows often form as far off center as the equator, they somehow always form along a meridian through the animal pole. This seems difficult to explain by the orthodox, diastral model. Rather, it suggests that the cleavage furrows in Xenopus--and perhaps in animal cells quite generally--are directly induced by a diastema or telophase disc rather than by the asters.

Ooplasmic Segregation in the Medaka (Oryzias latipes) Egg.

Using time-lapse video microscopy, we found that ooplasmic inclusions in the fertilized medaka egg displayed two types of movement during ooplasmic segregation. The first manifested itself as the movement of many inclusions (diameter = 1.5-11 µm) toward the animal pole at about 2.2 µm min-1; this type of movement appeared to be streaming. The second type of movement was faster (about 44 µm min-1) and saltatory; inclusions displaying this type of movement were smaller (diameter ≤1.0 µm) and moved toward the vegetal pole. The movement of oil droplets toward the vegetal pole of the egg may represent a third type of motion. All these movements began only after a strong contraction of the ooplasm toward the animal pole, which at 25°C began 10-12 min after fertilization and <3 min after formation of the second polar body. In eggs treated with microtubule poisons--colchicine, colcemid, or nocodazole--oil droplets did not move toward the vegetal pole, saltatory motion toward the vegetal pole was absent, and the growth of the blastodisc was slowed. Eggs treated with ß-lumicolchicine, an inactive derivative of colchicine, showed normal movements. Colchicine, while not inhibiting formation of the second polar body, did inhibit pronuclear migration. These results suggest that microtubules are involved in the movement of some ooplasmic inclusions, including oil droplets, toward the vegetal pole; the movement of ooplasmic inclusions toward the animal pole; and pronuclear migration.

High Calcium Zones at the Poles of Developing Medaka Eggs.

We have injected medaka fish zygotes with recombinant aequorin and visualized the resulting patterns of luminescence to reveal patterns of free calcium during early development. We have co-injected fluorescein-labeled aequorin to correct for nonuniformities in aequorin (as opposed to calcium) distributions by visualizing the resulting patterns of fluorescence as opposed to luminescence. We have also coinjected a calcium buffer to facilitate calcium diffusion, dissipate apparent calcium gradients, and thus confirm their reality. An exploratory study shows zones of elevated free calcium at the vegetal as well as the animal pole during the first day of development and thus up to the beginning of gastrulation. A closer study during the first 6 h, and thus through ooplasmic segregation and early cleavage, shows a steady zone of high calcium at the vegetal pole and a slowly oscillating one at the animal pole. The latter is particularly strong during ooplasmic segregation and cytokinesis. This report contains the first unambiguous evidence of relatively steady zones of high cytosolic calcium during the development of an animal egg.

Slow calcium waves accompany cytokinesis in medaka fish eggs.

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.

Electrical currents associated with rhythmic contractions of the blastoderm of the medaka, Oryzias latipes.

1. We used a vibrating probe to measure extracellular electrical currents near the surface of dechorionated Oryzias latipes eggs as contraction waves moved slowly across the blastoderm. 2. Although we found no detectable current outside dechorionated embryos, we recorded large current pulses near the edge of wounds made in the surface of the blastoderm. 3. The maximum net inward current--or in some cases, the least net outward current--correlated temporally with the contraction of cells near the edge of the wound. 4. The current pulses were superimposed on steady currents of variable magnitude and polarity. 5. We discuss possible mechanisms for the initiation and propagation of the contraction wave.

Pacemaker region in a rhythmically contracting embryonic epithelium, the enveloping layer of Oryzias latipes, a teleost.

The primary objectives of this study were to determine the embryonic stage at which the Oryzias latipes enveloping layer (EVL) begins to contract rhythmically, and to determine where these contractions arise within the EVL. Using time-lapse video recording, we showed that the contractions begin at stage 14 (the stage of the embryonic shield) and arise in the ventral region of the EVL, which is centered at 180 degrees longitude from the embryonic shield. We have called this the pacemaker region for the contractions. Using fluorescein diacetate as a vital stain, we showed that the ventral region of the EVL continues to act as a pacemaker even after the EVL is detached from the rest of the egg. Rhythmic contractile activity ceased when we removed a group of about 130 cells--10% of the total EVL--from the pacemaker region; comparably large wounds elsewhere had no effect on the contractions. When we cut detached EVLs into ten pieces, only 2.4 +/- 1.8 (mean +/- SD, N = 11) of them contracted rhythmically, even though a considerably larger proportion of the EVL cells participate in the contractions in undisturbed blastoderms. We conclude that the pacemaker cells are necessary for rhythmic contractile activity and that cells outside this region do not contract spontaneously. The contractile waves are propagated at a velocity of 14-54 microns sec-1. This value, which is two to three orders of magnitude slower than the propagation of epithelial action potentials, is similar to the rate of propagation of waves of increased cytosolic Ca2+ in other systems. We propose that the medaka EVL is a good system in which to study certain aspects of epithelial morphogenesis.