/ . Embryol. cxp. Morph., Vol. 14, Part 1, pp. 15-24, August 1965
Printed in Great Britain
Cell numbers during cleavage of the zebra fish egg
by
A. W. MARRABLE 1
From the School of Veterinary Science, University of Bristol
paper reports a study of the timing of early cleavage divisions in the
zebra fish and of the changes that occur during cleavage in cell size and number.
THIS
MATERIAL
The eggs of the zebra fish Brachydanio rerio (Hamilton-Buchanan) were used
in these studies. Roosen-Runge (1936, 1938, 1939), Marrable (1959,1962a) and
others have described various aspects of the biology of this fish when maintained
in the laboratory. It is a freshwater tropical teleost, the females laying nonsticky, non-pigmented demersal eggs which undergo a regular and predictable
pattern of cleavage; fertilization is external and the diameter of the spheroidal
chorion enclosing each egg is about 800 /x. Breeding is normally promiscuous
but when monogamy is imposed hundreds of fertile eggs can be obtained.
Development is rapid: at 27-25±0-5°C. the first cleavage is 30 min. after
fertilization; there are 32 cells after 90 min. and gastrulation takes place after
about 4 hr.
METHODS
Newly fertilized eggs from monogamous pairs of fish were maintained at
27-25±0-5°C. in an observation chamber mounted on a stereoscopic microscope. Each sample comprised from six to ten eggs; each egg was identifiable
by its location in the chamber.
Cleavage rate
Under the conditions of these experiments it was not possible to record the
moments of fertilization and the earliest events that could be timed were the
appearances of the first cleavage planes. As cleavage proceeded the times of
divisions were recorded but after the 32-cell stage no reliable observations
could be made by this method because of the diminution in the size of the
cells and the onset of their stratification.
The durations of the 2-, 4-, 8- and 16-cell stages were obtained for each egg.
1
Author's address: Department of Veterinary Anatomy, University of Bristol, Park Row,
Bristol 1, U.K.
16
A. W. MARRABLE
These values provided an index of the initial rate of development of each egg
and were compared by analysis of variance.
Nuclear number
After the 32-cell stage the eggs developed until each was fixed in Bouin's
fluid at a predetermined time. The chorion and most of the yolk were removed
and the blastoderm prepared for microtomy. Serial sections of twelve selected
eggs were cut at 7 or 8 yt, and stained with Heidenhain's haemotoxylin. Each
section on the completed slides was given a serial number and the nuclei and
nuclear fragments were counted in every section of each egg. The total count
for each egg was corrected for fragmentation by the methods described by
Marrable (19626). Separate tallies were kept for cells in mitosis and for nuclei
in the syncytial periblast; a percentage mitotic index was calculated for each
eggSince division is not instantaneous the following criteria were used to decide
at what stage one cell becomes two:
(1) The appearance of a cytoplasmic furrow at the outside of a cell. This
was used most when studying the living egg.
(2) The presence of a cytoplasmic partition, complete or incomplete, between
adjacent nuclei seen in stained sections.
(3) The presence in telophase of chromosomal vesicles containing small
central cavities, their internal diameters exceeding the thicknesses of their
walls.
Although decisions were frequently made using but one rule many cases were
observed when both (2) and (3) were fulfilled and a few when all three were
satisfied. It was therefore assumed that the criteria were of equal weight or that,
at least, the discrepancies involved were small compared with other errors.
Nuclear size
The principles governing the relations between the true diameter of the
nuclei and the diameters of their fragments have been discussed by Marrable
(19626). Each measured nuclear object was chosen as follows: a number was
picked from a random collection (Fisher & Yates, 1953) and the microscope
focused on the section bearing that number. Using a second random table and
a numbered graticule in the eye-piece, a nuclear fragment was similarly selected.
The maximum and minimum diameters of the fragment were measured with an
eyepiece micrometer; the mean of the measurements was taken as the fragment
diameter and was converted to microns.
The measurements for each egg were allotted to size-classes differing by
0-5 ix. The frequency distributions obtained were displayed in a composite
histogram (Text-fig. 1) showing the change in nuclear fragment diameter during
Cell numbers during cleavage in the zebra fish
17
the latter part of cleavage. The histogram is confined to those nuclei possessing
visible nuclear membranes.
0
Nuclear fragment diameters (microns)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
i
i
i i i
i i i
i i i i i i i i i
Age from fertilization
(mins.)
1153
166
n
186
199
010
204
5H
C/J
•S o.
S 10'
215
>^ 5U
g 0.
229
015"
105"
239
o248
1050151050"
0
-•263
l.
I.P
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Nuclear fragment diameters (microns)
TEXT-FIG. 1. Histograms showing the shift in distribution of nuclear fragment diameters
during the latter part of cleavage.
Cell size
The median section of each
final magnification of x 467.
chosen for measurement; the
and the nucleus in the centre
2
egg was photographed and prints were made at a
Each print was examined and ten cell-sections
uninterrupted cell boundary had to be traceable
of the cytoplasm. Selected cells were numbered
18
A. W. MARRABLE
and their areas measured on the print with a planimeter. Areas are magnified
as the square of linear magnification and the measurements were corrected to
give the sectional areas of the cells in square microns.
RESULTS
Sixty eggs were watched in nine experiments. The development of one egg
was so abnormal that it was excluded from further consideration; twelve eggs
did not cleave and possibly were not fertilized. As an index of cleavage rate for
each experiment, the mean duration of observations was calculated with its
standard error. These statistics are shown with a summary of the analysis of
variance in Table 1.
TABLE 1
Summary of observations on the living eggs
Experiment
No. of
eggs
1
2
3
4
5
6
7
8
9
3
6
5
2
5
4
7
9
6
Mean duration
of stages
(min.)
15-1
16-3
14-9
15-3
15-9
15-7
14-9
15-8
14-6
Variation between:
S.E.
(min.)
±008
±009
Eggs
Stages
±0-05
±0-53
±005
±006
±008
±003
±0-15
S.E. = Standard error of mean.
- = Not significant.
+ = Significant at the level, p< 0-05.
+ + = Significant at the level, p< 0 01.
+ + + = Significant at the level, p< 0 001.
The outstanding result is that in no batch were any significant differences
demonstrated between the rates of cleavage of individual eggs. In experiments
2, 4 and 9 there were, however, significant differences between the durations of
the different stages; the detailed records of these experiments suggest that when
there was an unusually short or long intercleavage period it may have been
compensated for in other stages.
There are significant differences in cleavage rate between some batches;
for instance using a /-test a difference was found between the 16-cell stages of
experiments 1 and 2 (p < 0-01). The tabulated means with their standard errors
permit further paired comparisons when necessary.
The results of the counting and of the nuclear and cell measurements are
shown in Text-fig. 1 and Table 2.
Cell numbers during cleavage in the zebra fish
19
TABLE 2
Summary of information on cell number and size
Egg
B02/5
1C
2A
2B
ID
2C
2D
2E
9C
IE
3A
8W
8Z
9F
5C
Age
(min.)
30-44
115
120
139
143
153
166
186
199
204
215
229
239
248
263
Total
nuclear
number
2
64
—
174
—
197
412
794
1325
1370
1725
2296
2781
3532
3880
M
Nuclei
in mitosis
2
—
—
—
—
165
219
42
68
275
319
257
301
232
291
M.I.
100
—
—
—
—
83-8
53-2
5-3
51
20-7
18-5
11-2
10-8
6-6
7-5
Mean diameter
Mean
Nuclei in
nuclear
cross-area
periblast fragments (JU.) of cells (/A2)
—
—
39956
—
—
—
—
3516
—
1726
—
—
2345
—
12-1
1721
26
10-3
835
36
110
1024
52
10-2
638
66
9-9
767
67
422
8-5
112
8-7
340
124
8-5
386
194
8-3
330
271
259
7-4
Number <Df cells in mitosis ,,
TO
Total number of cells
Age = Minutes from fertilization.
DISCUSSION
From observations on the living eggs it was concluded that the rates of
development were sufficiently close to justify the pooling of data. Table 3
shows the initial cleavage data of the twelve eggs which were used, when fixed,
to establish cell numbers in older blastoderms. The mean duration of each
stage has been used in constructing the part of the graph from 30 to 92 min.
(Text-fig. 2). It was not possible to observe fertilization, so the duration of the
1 -cell stage (Text-fig. 2: 0-30 min.) was based on the observations of RoosenRunge (1938). As in some other species, this stage is about twice as long as the
durations of the succeeding early cleavage stages; a possible explanation of this
is discussed by Swann (1957). The graph was completed from 92 to 263 min.
using the nuclear counts made on the twelve eggs after fixation (see Table 2).
The jy-axis gives the logarithm of the cell number but the x-axis (time) is an
arithmetic scale.
The step-like beginning of the graph is due to synchronous cell division.
The initial cleavage rate is departed from at the 197-cell stage after 153 min.
at 27-25°C. and the overall rate declines at least to the beginning of gastrulation. Deviation from the initial rate is gradual but the effect on cell numbers is
cumulative; by 229 min. when there are actually 2296 cells present, there would
have been about 10,000 had the initial rate been maintained.
The observations from 166 min. onward might appear to lie on a smooth
connecting curve. However, this impression must be rejected because it is
20
A. W. MARRABLE
improbable that the synchrony shown up to at least 90 min. is lost suddenly.
It is likely that one cell or a group of cells will lag behind the main wave of
divisions and evidence for this happening has been seen at 115 min. in the 64-cell
stage, where cells have been classified by their mitotic phase: metaphase 1,
anaphase 8, late anaphase 51, telophase 2, unclassified 2. If the initial rate is
not exceeded and the cells in telophase are the ones that have most nearly
maintained it, then the main wave as a whole has been delayed. On this hypothesis it is to be expected that successive waves of cell divisions will possess
TABLE3
Duration of early cleavage stages of those eggs studied later in the fixed state
(time in minutes)
Number of cells in stage
t
Egg
1C
2B
2C
2D
2E
9C
IE
3A
8W
8Z
9F
5C
2
10
14
8
15
15
17
15
15
15
15
15
15
15
14
15
16
16
19
16
19
17
15
15
15
17
16
15
18
14-5
16-9
15-1
16-5
14
16
15
14
16
14
14
(15)
(16)
(16)
Means
for stages
\
4
14
16
18
17
17
16
16
15
16
15
16
16
Grand Mean = 15-5.
Means
for eggs
14-75
16-50
16-50
16-25
16-25
15 00
15-00
15-00
1600
15-50
13-75
15-75
( ) = Estimated values.
similar features but with further development. Eventually the leading cells of
one wave will divide at the same time as the laggards of the next and the steps
of the graph will fuse into a complex waveform.
Many observations of cell numbers would be needed to establish the detailed
form of such a function but it is probable that certain features are sufficiently
prominent to be identifiable in my data. For instance such a waveform consists
of periods of high rate of increase alternating with gentle slopes of relative
stability. The cell numbers at 139 (egg 2B) and 153 min. (egg 2C) differ by 23
and are probably observations on the same near-horizontal part of the graph;
their ages differed by 14 min. suggesting the observations were at the beginning
and end of the same interphase. Upon examination of 2B the majority of cells
were seen in earliest interphase with chromosomal vesicles and persistent stem
bodies. In 2C many cells were in metaphase and a few were in anaphase or
early telophase; division was imminent. If we neglect the possibility of cell
I
I
I
I
I
I
60
1
I
I
1
i
I
I
I
i
I
I
120
180
Age from fertilization (mins.)
I
I
I
I
I
I
I
240
I
I
I
ioo
TEXT-HG. 2. The zebra fish: cell number and size as functions of time. Based on the observations in Tables 1, 2 and 3.
Total cell number • ; cell cross-area O (sample mean); nuclear fragment diameter + (sample mean).
0
=1000
10=
u
in
in
°2
V
r
=10,000
100=
27-25 A 0-5°C
pl00,000\
Temperature
1000 =
Cell number
10,000
C5
22
A. W. MARRABLE
growth between cleavages we shall expect the cell size to be similar in these two
eggs. It is; egg 2B having a mean cross sectional area of 1726 /A2 and 2C 1721 /u,2.
These facts suggest that a stable period connects the observations on eggs
2B and 2C.
Further, when the time of the beginning of the 32-cell stage (mean value
92 min.) is subtracted from the age of egg 2B (139 min.) the balance (47 min.)
is approximately a whole number multiple of the mean duration of the early
stages (15-5 min.). This permits the assumption that there had been two waves
of mitoses between the 32-cell stage and that of egg 2B. The times of division
have been estimated at approximately 108 and 124 min. giving three plateaux
of 15 -7 min. duration (the latter value obtained by dividing 47 min. by 3). The
height of the second estimated plateau has been put at the 64-cell stage as
there is a count of cells in egg 1C at 115 min. By halving the ordinate distance
between 1C and 2B the third plateau is estimated at cell number 117.
It is thought that there is a similar near-horizontal part of the graph located
between 199 and 204 min. Egg 9C, fixed at the beginning of this period, has
1325 cells whilst IE fixed at the end has 1370; their cells have similar mean
cross-sectional areas, 638 and 767 JX1 respectively.
Cell and nuclear sizes have been plotted on Text-fig. 2 for comparisons. The
time scale remains the same but the large range of cross-sectional areas required
condensing and a logarithmic y-axis was used; for the nuclear diameters an
arithmetic scale sufficed. The expected diminution in cell and nuclear size can
be seen, but the downward trends show short term irregularities. For cell-size
it is probable that in early and intermediate stages (say up to about 160 min.)
cells that had major and minor axes were non-randomly orientated and so their
cross-sectional areas varied with the plane of section. This effect and small
samples of measurements may account for the minor fluctuations and an
additional contribution may come from the growth of cells between divisions.
In fact, prophase nuclei were visibly larger than those in post-telophase and the
predominant nuclear phase in an egg will bias the value of mean nuclear size.
SUMMARY
1. The early cleavage rates of sixty living eggs of the zebra fish, Brachydanio
rerio (Hamilton-Buchanan) have been determined within the temperature range
27-25±0-5°C.
2. It was found that the cleavage rates were sufficiently similar to justify
pooling data obtained from different eggs. The mean inter-cleavage time was
15-5 min. during the period studied (2 to 32 cells inclusive).
3. Older eggs were fixed at pre-selected times in later cleavage. Twelve eggs
were prepared histologically and the nuclei counted in each. Measurements
were made of nuclear fragment diameters and cell cross-sectional areas.
4. Many of the data obtained (Tables 1, 2 and 3) were used to construct an
Cell numbers during cleavage in the zebra
fish
23
empirical graph (Text-fig. 2) showing total cell number during cleavage as a
function of the time after fertilization. Data on nuclear and cell size have been
similarly presented.
5. The end of cleavage and the beginning of gastrulation occur about 4 hr.
after fertilization at this temperature. The egg examined nearest to this time
contained 3880 nuclei, 259 of which were in the periblastic syncytium; the sample
of cell cross-sectional areas had a mean of 271 /u,2 and the nuclear fragment
distribution a mean of 7-4 /x.
RESUME
Nombre de blastomeres pendant la segmentation de Voeufdupoisson Brachydanio
rerio.
1. On a determine le rythme de segmentation precoce de 60 oeufs vivants de
Brachydanio rerio (Hamilton-Buchanan) dans l'intervalle de temperature de
27-25 ± 0,5° C.
2. On a trouve que les rythmes de segmentation etaient assez semblables
entre eux pour justifier la reunion des resultats obtenus a partir de differents
oeufs. L'intervalle de temps moyen entre deux clivages etait de 15 min. 30 sec.
pendant la periode etudi6e (de deux a trente-deux blastomeres inclus).
3. Les oeufs plus ages ont ete fixes a des periodes choisies a l'avance, au cours
des clivages tardifs. Douze oeufs ont ete traites pour l'etude histologique et les
noyaux y ont ete comptes. On a mesure des diametres de fragments nucleaires
et des surfaces de cellules coupees transversalement.
4. Parmi les resultats obtenus (Tableaux 1, 2 et 3), un grand nombre ont
ete utilises pour construire un graphique empirique (Fig. 2 dans le texte)
montrant la variation du nombre total de cellules au cours de la segmentation
en fonction du temps ecoule depuis la fecondation. Les resultats concernant la
taille des noyaux et des cellules ont ete presentes de maniere semblable.
5. La fin de la segmentation et le debut de la gastrulation surviennent 4 hr.
environ apres la fecondation a cette temperature. L'oeuf examine qui correspondait le plus etroitement a ce laps de temps, contenait 3880 noyaux, parmi
lesquels 259 se trouvaient dans le syncitium periblastique. L'echantillon de
surface cellulaire en section transversale mesurait en moyenne 271 fx2 et la
repartition des diametres de fragments de noyaux avait 7,4 //. en moyenne.
REFERENCES
R. A. & YATES, F. (1953). Statistical Tables for Biological, Agricultural and Medical
Research. Oliver and Boyd: Edinburgh and London.
MARRABLE, A. W. (1959). Variations in early cleavage of the zebra fish. Nature, Lond. 184,
1160-1.
MARRABLE, A. W. (1962a). The initial development of the zebra fish: a study of cell proliferation. Ph.D. thesis: University of Bristol.
FISHER,
24
A. W. MARRABLE
A. W. (19626). The counting of cells and nuclei in microtome sections. Q. J.
microsc. Sci. 103, 331-47.
ROOSEN-RUNGE, E. C. (1936). Furchung und Primitiventwicklung von Brachydanio rerio.
Anat. Anz. 81, 297-301.
ROOSEN-RUNGE, E. C. (1938). On the early development—bipolar differentiation and cleavage
—of the zebra fish, Brachydanio rerio. Biol. Bull. mar. biol. Lab., Woods Hole, 75,
119-33.
ROOSEN-RUNGE, E. C. (1939). Karyokinesis during cleavage of the zebrafish,Brachydanio
rerio. Biol. Bull. mar. biol. Lab., Woods Hole, 77, 79-91.
SWANN, M. M. (1957). The control of cell division: a review. Cancer Res. 17, 727-57.
MARRABLE,
{Manuscript received 23rd January 1965)