J. Embryol. exp. Morph. Vol. 19, 2, pp. 203-15, April 1968
With 7 plates
Printed in Great Britain
203
Yolk distribution
and utililization during early development of a
teleost embryo {Brachydanio rerio)
By ROBERT J. THOMAS 1
From the Department of Biochemistry and Biophysics,
Iowa State University
The work of Oppenheimer (1934, 1936) and Tung (1955, see also Devillers,
1961) suggests that an embryo such as Fundulus, a marine teleost, explanted at
early cleavage (two-cell stage) is more dependent upon the amount of periblast
yolk included in an explant than is an embryo that is more advanced prior to
explanation. In other words, 'if it (the embryo) is explanted before a so-called
critical stage, the blastoderm turns into a hyperblastula (a non-differentiated
mass of cells); if it is explanted after that, it undergoes differentiation. The
critical stage corresponds to eight blastomeres in Carassius, thirty-two in
Fundulus, and a young blastula in Salmo' (Devillers, 1961, p. 391). The zebrafish,
Brachydanio rerio, with a smaller egg than these fish, has not been characterized
by explanation. Devillers, in summary, states that 'these results mean that a
substance indispensable for differentiation exists in the yolk sphere' (p. 392).
He continues,' On the other hand, how this hypothetical material may reach the
blastoderm needs to be explained. The base of the blastoderm is in direct contact
with the periblast in the early stages; later on, this syncytium 'buds' off
blastomeres that add themselves to the embryonic disc' (p. 397). He then asks
the question 'but then how can one explain that in later stages the syncytium
can still impose an orientation on the germ? Are diffusing organizing substances
involved?' (p. 397).
Several questions may be further suggested from Devillers' initial question.
After the first horizontal cleavage, is more yolk being added to the cells of the
blastoderm? How is the yolk added to the cells of the blastoderm? What is in
the yolk that may be the indispensable substance suggested by Devillers ? This
study will attempt to answer these questions.
1
Author's address: Department of Biological Sciences, State University of New York at
Albany, New York 12203, U.S.A.
204
R. J. THOMAS
MATERIALS AND METHODS
The developing embryo
Mature zebrafish, Brachydanio rerio (Hamilton-Buchanan), were obtained
from commercial hatcheries,. In the laboratory, the fish were kept in balanced
aquaria at 25 °C with a day-period of 14 h (tungsten illumination). Both live
brine shrimp (Artemia salina), hatched in the laboratory from dry eggs, and dry
fishfood were fed to the fish daily.
For breeding, a gravid female and several mature males were confined in a
plastic breeding trap kept at 26 °C in an aquarium without sand or vegetation.
Breeding usually occurred, if at all, within the first hour of 'day' illumination
(Hisaoka & Firlit, 1962 a). Fifty to 300 closely developing eggs were found in the
bottom of the spawning tank and placed in a medium-sized dish for observation
and use. The use of very closely developing populations from a given batch of
eggs was accomplished by further selection of subspawns chosen by taking eggs
which progress from the 1-cell to a 2-cell stage within a 60 s period. The developmental stages of B. rerio enumerated and timed by Hisaoka, Ott & Marchese
(1957) and Hisaoka & Battle (1958) for 26 °C. incubation were used. Embryos
from early high blastula (stage 8) to closure of the blastopore (stage 17) were
fixed for studies of normal morphology or were exposed to experimental milieu
and subsequently fixed.
Experimental treatments
Developing embryos at selected stages were exposed to either 49-5 % deuterium
oxide or 10~ 3 M colchicine (Calbiochem) in aquarium water for various time
periods. Parallel subspawn samples of eggs with chorions torn to provide freer
access of solutions were exposed to the abnormal milieu to determine the
availability of the milieu to the embryo. For the materials used, the chorion did
not hinder availability to the embryo. In all cases, some of the controls were
permitted to hatch to test the viability of the spawn.
Fixation of embryo
During the study of normal events, eggs were fixed for 30 min with 1 %
osmium tetroxide with either 3 x 10~ 3 M or 10~ 2 M calcium chloride buffered with
veronal acetate at pH 7-5; or 1 % hydroxyadipaldehyde buffered with phosphate
at pH 7-5, rinsed in phosphate buffer at pH 7-5 and postfixed in 1 % osmium
tetroxide buffered with phosphate at pH 7-5 (Sabatini, Bensch & Barnett, 1963);
or with 1 % hydroxyadipaldehyde buffered with sodium cacodylate at pH 7-6,
rinsed in sodium cacodylate buffer at pH 7-6 and postfixed in 1 % osmium
tetroxide buffered with veronal acetate at pH 7-6. Experimentally treated
material was fixed only in 1 % osmium tetroxide with 10~ 2 M calcium chloride
buffered with veronal acetate at pH 7-5. All fixatives were adjusted to a final
osmolarity of 172 m-osmoles (calculated Powell, Philpott & Maser, 1964;
Teleost yolk
205
Maser, Powell & Philpott, 1967). The fixed eggs were dehydrated through an
ascending series of graded ethanols. Eggs were embedded in Epon resin (Luft,
1961). Thick sections were hand-cut with razor blades or with glass knives on a
Reichert Ultramicrotome OMU-2 for preliminary observations and correlation
of stage morphology to the in vivo observations just prior to fixation. Hisaoka &
Firlit's study (1960) of sectioned paraffin-embedded B. rerio eggs was used as a
basis of comparison. Thick sections were observed and photographed under
phase-contrast optics. Selected embryos were sectioned at 40-65 m/i with an
LKB Ultrotome with a Dupont diamond knife or a Reichert Ultramicrotome
OMU-2 with glass knives and mounted on 75 x 300-mesh unfilmed copper grids.
All sections for electron microscopy were stained with lead citrate (Reynolds,
1963) prior to observation on an RCA EMU-3F electron microscope operated at
either 50 or 100 kV.
RESULTS
Yolk distribution: normal
At early high blastula (stage 9) the blastomeres are situated on the yolk mass
(Plate 1, fig. A). Yolk, as round opaque particles, is visible at low magnifications
with phase-contrast optics and is located at the periphery of all cells (Plate 1,
fig. A). After the first horizontal cleavage, large yolk particles (4/A maximum
diameter) as well as yolk particles equal in size or smaller than the 0-5 /i mitochondria are found in the cytoplasm.
Below the blastomeres in the periblast, cytoplasm that is contiguous with the
yolk mass (Plate 2, fig. A) contains large closely packed particles of yolk.
Ribosome-like particles can be seen in high numbers in the blastomeres and
between the yolk particles of the periblast. Each yolk particle is surrounded
by a membrane (M) if an appropriate cross-section is observed (Plate 2, fig. B).
The interior of the yolk particle appears uniform under phase-contrast optics
Plate 1, fig. A). At the electron-microscopic level of observation, membranous
vesicles (V) can occasionally be seen within the yolk (Plate 2, fig. B).
In cells of the late high blastula, yolk particles are located peripherally to the
nucleus or the mitotic apparatus (Plate 1, fig. B). The number of yolk particles
in cells near the periblast is higher than in cells toward the surface of the embryo.
The periblast contains a large variety of yolk particle sizes (Plate 3, fig. A) as
compared to those in the periblast at early high blastula (Plate 2, fig. A). In
fortuitous section, contact of the blastomeres (B) with the periblast syncytium
(P) shows no membrane-membrane contact but rather a row of vesicles separating
periblast from blastomeres (Plate 3, fig. B).
Yolk particles are found to be more numerous in the cells close to the
periblast from late high blastula (Plate 1, fig. C) to the one-third epiboly stage
(Plate 1, fig. F). Various sizes of yolk particles are in the periblast syncytium
in these same stages. However at one-half epiboly (Plate 1,fig.G), only randomly
scattered cells have definite yolk particles. The periblast yolk of the one-half
14
JEEM 19
206
R. J. THOMAS
epiboly embryo (Plate 1,fig.G) appears similar to that of the early high blastula
embryo (Plate 1, fig. A). Various-sized yolk particles present in the periblast of
embryos from late high blastula to one-third epiboly stage are absent at the onehalf epiboly stage.
Throughout the entire development period from late high blastula to one-half
epiboly stage, yolk particles that were first seen in all cells of the early high
blastula embryo are missing from cells furthest from the periblast.
Yolk distribution: experimental
Eggs at the eight-cell stage were exposed to either 10~3M colchicine or 49-5 %
deuterium oxide. In a period of 3 h the control embryos progressed to the flat/
very-late blastula stage (Plate 4, fig. A). Their gross morphology is indistinguishable from other material at the same stage used in this study (Plate 1,figs.C, D).
Colchicine exposure results in immediate mitotic arrest (Plate 4, fig. B).
Cleavage did not occur in the exposed eggs. Some yolk particles are seen
within the cells but are fewer and smaller than those seen in an early high
blastula embryo. The periblast is enlarged and small yolk particles are near the
yolk mass (Plate 4, fig. B).
After 3 h exposure to deuterium oxide, the gross morphology of the blastula
is similar to that of the flat/very-late blastula, but the cells have a diameter more
like late high/flat blastula embryos (compare Plate 4, fig. C to the normal cell
size in Plate 1, fig. C). Intercellular spaces are absent (Plate 4, fig. C). Many
mitotic figures with prominent poles are present. The quantity of yolk particles
in cells near the periblast appears the same as yolk particles in the control
embryos. The periblast is greatly enlarged with more small yolk particles in the
periblast than is seen in the control embryos (compare Plate 4, fig. C to control,
Plate 4, fig. A).
PLATE 1
Figs. A-G, x 125.
Fig. A. Early high blastula embryos have uniformly distributed yolk after bipolar differentiation. Note the absence of small yolk particles in the periblast syncytium.
Fig. B. Late high blastula embryos have smaller volume cells. Yolk particles in cells near the
periblast are more numerous than in cells away from the periblast.
Fig. C. Flat blastula embryos have smaller volume cells.
Fig. D. Very late blastula cells near the periblast contain the majority of yolk particles.
Mitotic figures in various phases are frequent.
Fig. E. At early gastula stage, the blastoderm is lower. Small yolk particles in the periblast and
in cells near the periblast are higher in number than in cells away from the periblast.
Fig. F. One-third epiboly stage embryos still show small yolk particles in the periblast and in
cells near the periblast.
Fig. G. At one-half epiboly stage, the marginal periblast has several prominent nuclei. Small
yolk particles in the periblast are absent. Only scattered cells above the periblast contain yolk
particles.
/. Embryol. exp. Morph., Vol. 19, Part 2
R. J. THOMAS
PLATE 1
facing p. 206
J. Embryol. exp. Morph., Vol. 19, Part 2
R. J. THOMAS
PLATE 2
J. Embryo!, exp. Morph., Vol. 19, Part 2
PLATE 3
Fig. A. Membrane-bound yolk particles within yolk particles are found in the periblast of
the late high blastula embryo. Hydroxyadipaldehyde buffered with sodium cacodylate fixation;
lead citrate stained, x 14600.
Fig. B. Blastomeres (B) are in 'intimate' contact with the periblast syncytium (P). There is
no definitive membrane-membrane separation but rather a row of vesicles. Hydroxyadipaldehyde buffered with sodium cacodylate fixation; lead citrate stained, x 8800.
R. J. THOMAS
J. Embryol. exp. Morph., Vol. 19, Part 2
R. J. THOMAS
PLATE 4
J. Embryol. exp. Morph., Vol. 19, Part 2
PLATE 5
Figs. A, C, E, x 125. Figs. B, D, F, x 320.
Figs. A, B. Control embryos after 12 h from late high blastula were all at one-half epiboly
stage. Small yolk particles are absent from the periblast cytoplasm. Nuclei are easily seen in
the periblast (arrow, fig. B).
Figs. C, D. Colchicine-exposed late high blastula embryos are arrested with cell diameters of
cells of the late high to flat blastula. Nuclei are absent. Note the almost complete absence of
yolk particles in cells near the periblast.
Figs. E, F. Deuterium oxide-exposed embryos have progressed to only the one-third epiboly
stage. Little yolk is present in the cells of the embryo.
R. J. THOMAS
J. Embryol. exp. Morph., Vol. 19, Part 2
. - . ,.
R. J. THOMAS
•*•
PLATE 6
J. Embryol. exp. Morph., Vol. 19, Part 2
R. J. THOMAS
PLATE 7
Teleost yolk
207
Six hours after initial exposure to the experimental milieu, the control embryos
(Plate 4, fig. A) are at one-third epiboly stage. The colchicine-exposed embryos
did not advance morphologically (not shown), and deuterium oxide-exposed
embryos (Plate 4, figs. E, F) showed cytolysis (breakdown of the cell membrane).
The tangential section of a deuterium oxide-exposed embryo shows numerous
periblast nuclei and broken cell membranes (arrow, Plate 4, fig. F). Deuterium
oxide exposure was terminated because of disintegration of the embryos.
As the control embryos continued to closure, 12 h after initial exposure of the
experimental embryos, the colchicine-exposed embryos remained morphologically unchanged. There was no indication of cell movement or epiboly.
Other eggs were initially exposed at a later developmental stage, early high
blastula (stage 9), to either colchicine or deuterium oxide for a period of 4 h.
During the period of exposure the control eggs progressed to the very late
blastula stage (Plate 4, fig. G).
Colchicine-exposed blastulae (Plate 4, fig. H) have cells of varying diameters,
and mitotic figures are absent. Many small yolk particles are found in the
periblast yet they are absent in the larger volume cells. Both intact nuclei and
free chromosomes are found throughout the blastula. The general cytoplasm
appears unchanged from the controls.
Deuterium oxide-exposed eggs progressed to the late high/flat blastula stage
as judged by cell diameters (Plate 4, fig. I). Intercellular spaces are absent. Yolk
particles are less frequent in all cells (Plate 4, fig. I, compared to control, Plate 4,
fig. G). However, the amount of yolk is less in the deuterium oxide-exposed cells
when compared to an embryo with a similar cell diameter (compare Plate 4,
fig. I, to Plate 1, figs. B, C).
Late high blastula embryos were exposed to either colchicine or deuterium
oxide for a period of 9 h. During the period of exposure, the control embryos
progressed to the one-half epiboly stage (Plate 5, figs. A, B). The marginal periblast is at the equator of the embryo and an enveloping layer covers the loose
cells of the embryo. Yolk particles are in scattered cells and the periblast does
not have any small yolk particles. Only periblast nuclei (arrow, Plate 5, fig. B)
and large yolk particles of the main yolk mass comprise the periblast.
Colchicine-exposed embryos treated as above (Plate, 5, figs. C, D) are
arrested at late high/flat blastula stage (stage 10/11; Plate 1, figs. B, C). Mitotic
figures are absent; cells are almost completely free of yolk particles.
PLATE 2
Fig. A. Periblast yolk of the early high blastula embryo is mainly large membrane-bound yolk
particles. Cytoplasm can be seen between some yolk particles in the periblast. Osmium
tetroxide with calcium fixation; lead citrate stained, x 5350.
Fig. B. At a higher magnification, the membranes (M) around the yolk particles are readily
apparent. Some yolk particles are within others but separated by their own membranes.
Vesicles (F) can be seen in some yolk particles. Osmium tetroxide with calcium fixation; lead
citrate stained, x 16600.
14-2
208
R. J. THOMAS
After 9 h exposure to deuterium oxide, an embryo exposed at late high blastula
stage has progressed only to the one-third epiboly stage (compare Plate 5, fig. E,
to Plate 1, fig. F). Cells contain significantly less yolk than a similarly advanced
embryo. The periblast contains a large number of small yolk particles.
Yolk fate
In the periblast some membrane-bound yolk particles nestled within others
(Plate 2,fig.A; Plate 3,fig.A) are suggestive of large yolk particle fragmentation.
In cells of all stages from late high blastula to one-half epiboly, some yolk
particles (0-5-1-5 fi in diameter) are seen with various ratios of yolk and centrally located membranous elements and ribosome-like particles (Plate 6, figs.
A, B, Plate 7, figs. A, B).
A cytoplasmic structure (4-7^ average diameter) found in a single cell
(Plate 7, fig. B) suggested the ultimate fate of yolk particles. The interior of this
complex structure contains numerous membranous vesicles and ribosome-like
particles. Separated from the cytoplasm by a membrane, a thin layer of material
similar in density and structure to a yolk particle (arrows, Plate 7, fig. B) is
delineated from the interior by a second membrane.
DISCUSSION
Bipolar differentiation, continuing into late cleavage stages of B. rerio
(Roosen-Runge, 1938), results in all cells having a complement of yolk particles.
The film of B. rerio cleavage by Hisaoka et al. (1957) shows streaming of material
toward the blastomeres until early blastula. At early high blastula only large
yolk particles (4 /i) and yolk particles the size of mitochondria or smaller were
carried into the blastomeres. The first horizontal cleavage of the fertilized egg
separates the blastomeres from the yolk within the periblast. The cell membrane
of the fertilized egg after the horizontal cleavage encompasses the periblast
nuclei and cytoplasm. The periblast cytoplasm contains membrane-bound yolk
particles and is contiguous with cytoplasm between yolk particles of the yolk
mass. The space between the yolk mass and the periblast syncitium observed in
Fundulus by Lentz & Trinkaus (1967) is also apparent in the phase micrographs
(Plate 1,figs.B-F). However, electron micrographs of the periblast cytoplasm and
yolk (Plate 2,fig.A) show no such space. The space present between the periblast
cytoplasm and the yolk mass should be considered' halo' from the phase-contrast
optical system.
We may assume that the yolk in any given cell will be utilized almost as readily
as in a neighboring cell. In this study, mitotically active cells with peripherally
located yolk were found on the central periblast of the yolk mass. Marrable's
data (1965) shows that B. rerio has a high mitotic index (83 %) and a uniform
distribution of mitosis at blastula stages. These data would suggest, excluding
the occurrence of yolk transfer from the periblast, that uniform distribution and
usage of intracellular yolk should be expected. However, yolk is diminished in
Teleost yolk
209
cells away from the periblast, and yolk particles are consistently seen in high
concentration in cells near the periblast.
Two possibilities may be considered for the occurrence of yolk in cells near
the periblast. Yolk particles were never found extracellularly. The direct transfer
of yolk by phagocytosis is excluded as a possible mechanism by Lentz &
Trinkaus (1967), who have reported that pinocytosis and phagocytosis are
'apparently absent in Fundulus\ The occurrence of vesicle boundaries (Plate 3,
fig. B) between blastomeres and periblast is suggestive of either transfer of
cytoplasm across the boundary or vesicle cleavage at the end of periblast mitosis.
In the blastoderm, both furrow cleavage and vesicle cleavage have been seen
with either osmium tetroxide with calcium or hydroxyadipaldehyde fixation.
Humphreys (1964) has shown similar vesicle cleavage boundaries with Mytilus
edulis polar bodies when polar body material is drawn into the cytoplasm of the
cell. Therefore, the other possible mechanism is transfer of yolk during mitosis
from the periblast syncytium.
Deuterium oxide, with a very large 'isotope effect', causes mitotic cells
immersed in deuterium oxide (70-96 %) to be immediately arrested regardless
of progress through the mitotic cycle (Gross & Spindel, 1960 a, b; Marsland &
Zimmerman, 1963, 1965). In this study, exposure of B. rerio embryos to concentrations of deuterium oxide (49-5 %), which would retard but not arrest
mitosis, allowed gross normal development of all embryos. Developmental
advancement, when cell diameters are compared (Marrable, 1965), was always
retarded; deuterium oxide exposure resulted in blastomeres that were swollen and
PLATE 4
Figs. A-E, G-I, x 125. Fig. F, x 320.
Fig. A. A flat to very late blastula embryo is developed from the eight-cell stage after 3 h.
Note the yolk particles in cells near the periblast. This embryo is a control for embryos shown
in fig. B and C.
Fig. B. An eight-cell embryo exposed to colchicine for 3 h does not advance developmentally.
Yolk particles are infrequent and smaller in size.
Fig. C. Embryos exposed to deuterium oxide for 3 h advance to late high to flat blastula
stages when cell size is compared to normally developed eggs. Intercellular spaces are absent
and the periblast is enlarged. Mitotic figures have prominent aster-like structures.
Fig. D. The control embryo for 6 h exposure to deuterium oxide proceeds to the one-third
epiboly stage.
Figs. E, F. A tangential section of an embryo exposed to deuterium oxide for 6 h shows broken
cell membranes (arrows, fig. F) and many nuclei in the periblast.
Fig. G. The control embryo for embryos exposed to colchicine or deuterium oxide at early
high blastula is at the very-late blastula stage after 4 h.
Fig. H. A colchicine-exposed embryo has a blastoderm of varying cell sizes. Yolk particles
are absent in the large cells near the free surface of the embryo.
Fig. I. Deuterium oxide-exposed embryos have progressed to the late high to flat blastula
stage embryos. Yolk in cells away from the periblast is diminished.
210
R. J. THOMAS
had reduced or missing intercellular spaces, and mitotic apparatuses that had
prominent astral regions. The effect of deuterium oxide is therefore similar to
that described by Gross & Spindel (1960 a, b).
Exposure of eight-cell embryos to deuterium oxide resulted in otherwise
normal development, even yolk utilization, until cytolysis occurred. Early high
blastula embryos exposed to deuterium oxide lagged developmentally and showed
fewer yolk particles in the blastoderm as compared with the control. Late high
blastula embryos proceeded to the one-third epiboly stage as the control embryos
proceeded to the one-half epiboly stage. Significantly, the cells of deuterium
oxide-exposed embryos did not contain as many yolk particles in the cells as in
similarly advanced embryos.
The developmental lag caused by exposure to deuterium oxide, although
possibly due to slowing of the mitotic cycle, would probably not cause cessation
of yolk utilization. If yolk utilization did continue in the blastomeres without
a contribution of yolk-containing cells to the blastoderm by the periblast, the
finding of cells with less yolk in the embryo would be expected. Indeed, the
deficiency of yolk in deuterium oxide-exposed eggs did occur. Therefore, the
conclusion may be advanced that mitosis from the periblast does occur and
results in yolk appearing in cells near the periblast. However, this hypothesis
cannot be confirmed by exposure of the embryos to deuterium oxide because of
cytolysis in some embryos exposed for 4 h or longer.
Some colchicine-exposed blastulae with a thick blastoderm are found with
large, surface cells but smaller deep cells. A surface cell is immediately exposed
to colchicine, but a cell deep within the blastoderm may be affected by colchicine
later due to penetration difficulties. This phenomenon of rapid exposure is most
apparent at the eight-cell stage when all cells are immediately exposed or at
late high blastula when the blastoderm is thin; early high blastulae have a
significantly thicker blastoderm which would present a problem of penetration.
In embryos which have cells that are considered rapidly exposed to colchicine,
no interphase nuclei are evident. However, in the early high blastula embryo
where exposure is considered variable because of penetration, interphase nuclei
are found.
Rapid arrest at mitosis by colchicine could be expected to affect any mitosis
that might occur from the periblast but not affect yolk utilization. Therefore
the question of yolk transfer from the periblast syncytium to the blastoderm can
be experimentally tested. If yolk is transferred to the cells of the blastoderm by
any means other than mitosis, yolk could be expected in some blastomeres in
spite of mitotic arrest. However, in embryos rapidly affected by colchicine, the
eight-cell and late high blastula embryos, yolk is relatively absent.
Early high blastulae exposed to colchicine, however, have reduced quantities
of yolk at the blastula surface where cells are arrested mitotically but normal
amounts of yolk in cells close to the periblast which are unaffected by colchicine.
If yolk is transferred by periblast mitosis, colchicine-arrested cells near the
Teleostyolk
211
surface should contain a reduced amount of yolk as compared to untreated cells
near the periblast.
However, if a yolk complement was given to cells during bipolar differentiation and subsequently utilized and new cells with yolk were formed from the
periblast, we would expect to find cells without yolk in untreated cells furthest
from the periblast and cells close to the periblast with a complement of yolk.
This situation is observed in normally developing embryos but is not seen in
embryos where mitosis has been uniformly stopped by colchicine.
As suggested by Oppenheimer (1934,1936), Tung (1955) and Devillers (1961),
some substance which carries information (or material) necessary for the
differentiation processes after a 'critical' stage is within the yolk. Identification
of the components of yolk should provide important information on the nature
of the 'yolk-substance' necessary for differentiation.
Yolk particles as seen in this study in one-third epiboly cells and earlier have
been observed initially to have only amorphous yolk material surrounded by a
unit membrane, then to have membranous elements and ribosome-like particles
in the center of membrane-bound yolk material, and to appear finally as a
highly complex particle appearing as cytoplasm surrounded by little yolk and a
unit membrane. These yolk particles containing various quantities and types of
inclusions could be arranged as suggested above in a sequential pattern of utilization. Bellairs (1958) has seen a similar, but more complex, sequential ordering of
yolk particles in the chick embryo at gastrulation and later. Strikingly, the final
member of the suggested sequence appears as an isolated portion of cytoplasm
surrounded by a thin layer of yolk and an external unit membrane. Thus the
proposal that a supply of maternal membranous elements and ribosomes be the
determining factor from the periblast necessary for permitting differentiation
at later development stages is a highly suggestive hypothesis.
Hisaoka & Firlit's work (19626) suggests the occurrence of RNA in a masked
form in yolk of the mature oocyte. After fertilization, RNA concentration
remains constant histochemically in all cells during cleavage and blastula
stages except for regional decreases during interphase and prophase (Hisaoka &
Firlit, 1961).
In Rana pipiens, Brown & Caston (1962) report the binding and masking of
exogenous isotope-labeled ribosomes to yolk in studies of in vitro protein
synthesis during early development. In R. pipiens, Karasaki (1963) notes the
PLATE 6
Fig. A. Yolk particles have a uniform granular appearance at high magnification except
where membranous vesicles are within the yolk. Osmium tetroxide with calcium fixation; lead
citrate stained, x 42100.
Fig. B. Yolk particles are seen with varying amounts of membranous material and ribosomelike particles. A sequence of these yolk particles may be used to describe the order of yolk
utilization. Osmium tetroxide with calcium fixation; lead citrate stained, x 13700.
212
R. J. THOMAS
decrease in yolk in the embryo, but he does not report ribosome-like particles
within Rana yolk platelets. Rounds & Flickinger (1958) conclude, however, from
their study of neural induction in R.pipiens that yolk breaks down in chordamesoderm with a subsequent release of RNA. Bellairs (1958) reports micro-particles
in yolk similar to the cytoplasmic micro-particles now known as ribosomes. In
the insect fat body, Locke & Collins (1965) have found isolation of membranes
and ribosomes in 'Protein + RNA granules' similar in appearance to B. rerio
yolk particles. Also in the sequestering insect fat body, granules fuse to form
larger 'fat' particles within cells; in contrast, in the periblast of B. rerio during
yolk utilization, large yolk particles appear to 'pinch, off' into smaller yolk
particles. These developing systems are considered diverse by Williams (1967)
and cannot be directly compared to teleost embryos.
The recent work of Perry (1967) on the morphological distinction between
glycogen and ribosomes shows the possibility of confusion between the two
molecules. The ribosome-like particles in this study are about 16-19 /i in
diameter, within the range for ribosomes characterized by Perry.
The conclusion is strongly suggested that membranous material and ribosomelike particles in the yolk of B. rerio originate maternally and are necessary for
differentiation. In addition, protein (yolk ?)-masked m-RNA as found recently
by Stavy & Gross (1967) in Echinoderm embryos might be also expected in
yolk of B. rerio.
Therefore, the several questions initially proposed from Devillers' question
may be answered as: yes, more yolk is added to the cells of the blastoderm by
periblast mitosis. The 'indispensable' substance contained in yolk necessary for
differentiation may be the maternal contribution of membranes and ribosomelike particles within yolk.
SUMMARY
1. Yolk is found evenly distributed in all cells of the blastoderm after bipolar
differentiation at the early high blastula stage. Small yolk particles are common
in the periblast until the one-third epiboly stage. Cells farther away from the
periblast during this period contain yolk particles which are decreased in number
and smaller in size. At one-third epiboly to one-half epiboly stage, small yolk
PLATE 7
Fig. A. The yolk particle in the center of the micrograph contains ribosome-like particles
similar to ribosomes of the cytoplasm. Osmium tetroxide with calcium fixation; lead citrate
stained, x 16500.
Fig. B. The ultimate fate of a yolk particle may be the structure which dominates this micrograph. Membrane-bound yolk, similar to yolk in a smaller particle (arrows) is separated from
the interior of the particle by a second membrane. The interior of the particle appears similar
to the cytoplasm outside the yolk particle. Osmium tetroxide with calcium fixation; lead
citrate stained, x 18700.
Teleostyolk
213
particles in the periblast are absent, and only scattered cells of the embryo
contain yolk. The hypothesis is suggested that mitosis from the periblast provides
new blastomeres with a yolk complement and is the mechanism of yolk transfer
to the blastoderm.
2. Exposure of B. rerio to 49-5% deuterium oxide solution causes a slowing
of normal development, a swelling of blastula cells, and ultimately cytolysis.
Although development and mitosis are slowed down, yolk utilization continues.
Cells away from the periblast contain smaller and fewer yolk particles than
cells of a control embryo with similar cell diameters. Mitosis from the periblast
is slowed down by deuterium oxide and could not then provide yolk-containing
cells to the blastoderm as rapidly. From this observation it is inferred that
mitosis from the periblast is providing cells to the blastula with a yolk complement.
3. Colchicine causes immediate cessation of mitosis and uniform developmental arrest if penetration is rapid. Arrested cells are found with much less yolk
than control cells of equal size. Cessation of mitosis and the absence of yolk
transfer is consistent with the hypothesis that cells with a yolk complement
originate from the periblast by mitosis.
4. Yolk particles found in B. rerio cells are seen initially to have only amorphous yolk material surrounded by a unit membrane, then to have membranous
elements and ribosomes in the center of membrane-bound yolk material, and
to appear finally as a highly complex particle that appears to have cytoplasm
surrounded by a little yolk and a unit membrane. The above order is suggested
as a sequential pattern of yolk utilization.
5. Yolk, besides providing soluble nutrients to the embryonic cells, contributes maternal membranous and ribosome-like material to the blastomeres.
RESUME
Repartition et utilisation du vitellus au cours des premiers stades
du developpement d'un embryon du Teleosteen Brachydanio rerio
1. Le vitellus se trouve reparti egalement entre toutes les cellules du blastoderme apres la differentiation bipolaire au premier stade de la blastula avance.
De petites particules vitellines se trouvent communement dans le periblaste
jusqu'au stade du tiers de l'epibolie. Des cellules plus eloignees du periblaste au
cours de cette periode contiennent des particules vitellines moins nombreuses et
plus petites. Du stade du tiers a celui de la moitie de l'epibolie, il n'y a pas de
petites particules de vitellus dans le periblaste et seules des cellules dispersees
dans l'embryon contiennent du vitellus. On suggere l'hypothese que les mitoses
a partir du periblaste fournissent de nouveaux blastomeres charges de vitellus
et qu'il s'agit la du mode de transfert du vitellus au blastoderme.
2. L'exposition de B. rerio a une solution a 49,5% d'eau lourde (D2O)
provoque un ralentissement du developpement normal, un gonrlement des
214
R. J. THOMAS
cellules de la blastula et finalement la cytolyse. Bien que le developpement et les
mitoses soient tres ralentis, l'utilisation du vitellus se poursuit. Les cellules
eloignees du periblaste contiennent des particules de vitellus plus petites et
moins nombreuses que celles des cellules d'un embryon temoin a diametres
cellulaires semblables. Les mitoses a partir du periblaste sont tres ralenties par
l'eau lourde et ne pourraient done pas fournir de cellules vitellines au blastoderme
aussi rapidement. De cette observation, on deduit que les mitoses du periblaste
fournissent a la blastula des cellules chargees de vitellus.
3. La colchicine fait cesser immediatement les mitoses et provoque un arret
general du developpement si sa penetration est rapide. Les cellules bloquees
renferment beaucoup moins de vitellus que les cellules temoins de taille egale.
L'arret des mitoses et l'absence de transfert de vitellus sont en accord avec l'hypothese selon laquelle les cellules chargees de vitellus prennent naissance par
mitose a partir du periblaste.
4. Les particules vitellines observees dans les cellules de B. rerio n'ont d'abord
qu'une substance amorphe entouree par une membrane unitaire, puis renferment des elements membranaires et des ribosomes au centre du materiel
vitellin limite par une membrane, et apparaissent finalement comme des particules
hautement complexes contenant du cytoplasme entoure par un peu de vitellus
et une membrane unitaire. On suggere que la succession precedente represente
le processus sequentiel d'utilisation du vitellus.
5. Le vitellus fournit des materiaux nutritifs solubles aux cellules embryonnaires, et apporte en outre aux blastomeres du materiel membranaire et du
materiel de type ribosomique tous deux d'origine maternelle.
This work was supported by Research Grants HD-2585 and HD-3762 to Dr L. Evans
Roth from the National Institute of Child Health and Human Development, United States
Public Health Service.
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