/. Embryol. exp. Morph. Vol. 40, pp. 277-283,1977
Printed in Great Britain © Company of Biologists Limited 1977
277
Differences in rotation-mediated
aggregation between wild-type and homozygous
Brachyury (T) cells
By K. O. YANAGISAWA 1 AND H. FUJIMOTO
From the Laboratory of Cell Biology,
Mitsubishi-Kasei Institute of Life Sciences, Tokyo
SUMMARY
Rotation-mediated aggregation was studied in dissociated cells from 9- and 10-day-old
(' 12-40 somite' stage) mouse embryos homozygous for the Brachyury (T) mutation. Average
diameter of the aggregates formed by wild-type cells increased up to the 27-somite stage.
In T/T cells, however, a completely different pattern was observed. The disappearance of
free cells and the process of aggregate formation were also followed. The possibility that
T/T cells have different adhesive properties from + / + cells is discussed.
INTRODUCTION
Dissociated embryonic cells can 'recognize' like from unlike and they sort
out to form tissue-specific groups. It is generally assumed that cell-surface
markers play an important role in cell-cell recognition and hence are essential
to differentiation (Moscona, 1962, 1965). Alleles at the Tit-locus of the mouse
may affect cell-cell recognition, since they are known to specify cell-surface components and to control early and basic processes in the organizational and
morphogenetic events occurring during embryogenesis (Bennett, 1958; Bennett,
Dunn & Artzt, 1976). The present work shows that TjT mutant cells have
different adhesiveness from + / + cells. The possibility that they also have
different recognition sites remains to be explored.
MATERIALS AND METHODS
Mice and embryos. T\ + mice were originally supplied by Dr Bennett. Homozygous mutant embryos were obtained from heterozygous matings (77 + x Tj+)
and control + / + embryos were obtained from matings of + / + parents segregated from T\ + xT/+ matings. Stages of T\T embryos were determined by the
number of somites of their normal litter-mates (Yanagisawa & Fujimoto, 1977).
1
Author's address: Laboratory of Cell Biology, Mitsubishi-Kasei Institute of Life Sciences,
11 Minamiooya, Machida-shi, Tokyo 194, Japan.
278
K. O. YANAGISAWA AND H. FUJIMOTO
Cell dissociation and culture. The trunks, heads and forelimb buds were isolated from the embryos. The excised tissue was cut into comparable sizes,
washed once with calcium-magnesium-free phosphate-buffered saline (PBS"),
then treated with 0-02 % trypsin for 1 min at 37 °C. Trypsin (Worthington, x 3
crystallized) was dissolved in PBS~ with 0-02% EDTA prior to each experiment. The reaction was stopped by adding 20 times volume of Eagle's minimum
essential medium supplemented with 10% heat-inactivated horse serum and
3 % chick embryo extract (MEMHC). The medium was discarded and washed
once more with the same volume of MEMHC. The desired volume of MEMHC
was added to the trypsinized tissue and incubated at 37 °C for 1 min. Then
the tissue was gently agitated three times with a Voltex mixer. Free cells thus
derived were collected in a tube and stored in an ice bath under an atmosphere
of 5 % CO2 in air. Storage of the cells in ice had no effect on aggregation. The
same procedure (PBS~ -* trypsin -* MEMHC -> MEMHC -+ MEMHC ->
mixer) was repeated on the remaining tissue until most of the cells had been
freed. The collected cells were centrifuged at 300 rev./min for 1 min to remove
undigested tissue fragments. The proportion of single cells was over 90%,
and the frequency of viable (nigrosin-excluding) cells was always over 95 %.
Cells were suspended in MEMHC at a desired concentration. One millilitre
of the suspension was cultured in a 5 ml siliconized Erlenmeyer flask on a
gyratory shaker at 85 rev./min (New Brunswick Co. Type G-24) at 37 °C.
Horse serum and chick embryo extract were purchased from Grand Island
Biological Company.
RESULTS
Dissociated + / + or T/T head or trunk cells were cultured on a gyratory
shaker for 16 h. The average diameter of the aggregates of + / + or T/T cells
is plotted against the number of somites in Fig. 1. The diameter of the + / +
aggregates increased as development proceeded, reaching its maximum value
around the 27-somite stage, with no further increase up to the 39-somite stage.
In TJT cells, the pattern was completely different. The diameter of the aggregates increased only from the '24-somite' to about the '27-somite' stage, and
thereafter declined. It was consistently lower than for + / + embryos. Similar
results were obtained with cells from the head and trunk regions of + / + or
T\T embryos (Fig. 1). Until about the '34-somite' stage, the head region of
TIT embryos resembles in gross morphology that of + / + embryos, yet the
T\T aggregates were smaller. The forelimb-buds of the T/T embryo start to
develop relatively normally at about the' 25-somite' stage and increase in volume
up to the '32-36-somite' stage. They develop into muscles and cartilages when
cultured in vitro (Fujimoto and Yanagisawa, unpublished observation; Ephrussi,
1935; Bennett, 1958), and show few abnormalities, yet the diameter of the
aggregates was again smaller than that of the + / + aggregates.
In both + / + and T/T cultures, the total volume of the aggregates roughly
Aggregation o/T/T cells
279
0-2
I 0-
16
20
24
28
Number of somites
32
36
40
Fig. 1. Average diameter of aggregates. + / + , trunk (O); + / + , head ( x ) ; T/T,
trunk ( # ) ; T/T, heads (A). Ordinate: average diameter of aggregates in mm.
Abscissa: number of the somites of the embryos from which the cells were dissociated. Stages of the T/T embryos were estimated from those of their normal
litter-mateS and the size of their heads.
280
K. O. YANAGISAWA AND H. FUJIMOTO
Table 1. Recovery of input cells in aggregates
Total volume*
8
(x 10> )
Number
Diameter Om)
t
Genotype
Stage
Cell
Aggregate
+ /+
TIT
TIT
35
'27'
'35'
ll-6±0-9
110±0-5
10-9 ±0-4
190±18
138±26
104 ±23
Aggregate
Input
formed
Cell Aggregate
cell
5
127
41
4-6
50xl0
184
3-8 xlO5
2-6
2-6
341
3-4
2-8
5-OxlO5
* -J-7T x (diameter of a single cell or an aggregate)3 multiplied by the number of input cells
or the aggregates formed.
Table 2. Initial cell number and the diameter of aggregates
Diameter (/*m)
number
6
20xl0 /ml
1-OxlO6
50xl05
3-0 xlO 5
1-OxlO5
5-0 xlO4
2-5 xlO4
+/+
464 ±268
204 ±16
190±18
198±26
182±12
180 ± 28
158 ±70
TIT
71 ±23
79±19
71 ±15
72 ±24
91 ±34
109 ±68
Cells were isolated from 10-day-old embryos (30-35 somites).
equals the total volume of single input cells (Table 1). In TIT cultures, however,
some cells attach to the culture vessel; the frequency of cells recovered in the
aggregates is over 83 % as calculated from the total volume of the aggregates.
As shown in Table 2, + / + cells (30-somite stage) formed aggregates with
similar diameters if the initial cell numbers were between 1 x 106 cells/ml and
5 x 104 cells/ml, and the T/T cells ('30-34-somite stage'), between 1 x 106 cells/
ml and 1 x 105 cells/ml. The 17% reduction in TIT cells is therefore not the
reason why they form smaller aggregates. The cross-sections of + / + and T\T
aggregates show that there are no differences in the frequency of dying cells,
suggesting that T\T cells are at least as viable as + / + cells during and after
formation of the aggregates.
Both the trunks and the heads used in the present experiment contain epidermal, mesodermal and neural cells. The ratio of neural to mesodermal cells
did not change with the stage or the genotype of the embryo.
Aggregates formed by dissociated trunk cells from + / + or TIT embryos
were cultured in Falcon plastic dishes in MEMHC. After 24 h, both mesodermal and neural cells were observed in cultures of both + / + and TfT
aggregates. In cultures of aggregates formed by cells from the forelimb-buds,
Aggregation o/T/T cells
o
30
60
90
281
120
Time (min)
Fig. 2. Initial mode of disappearance of free cells. One ml of + / + cells (O),
9-2 x 105/ml, or T/Tcells (x), 8-5 x 107ml, was cultured in MEMHC on a gyratory
shaker (85 rev./min) at 37 °C. Aliquots were taken at times indicated on the abscissa
and the number of single cells were counted and expressed as a percentage of
initial single cells.
282
K. O. YANAGISAWA AND H. FUJIMOTO
< < : • • ' . * .
D
•
_
^
i
Fig. 3. Process of aggregate formation. (A-E) + / + cells; (A) 1 h; (B) 3 h; (C) 5 h;
(D) 6 h; (E) 7 h; (F-J) TjTcells; (F) 1 h; (G) 3 h; (H) 5 h; (I) 6 h; (J) 7 h. x 40.
H
only mesodermal cells were observed. Differences in the cell types participating
in aggregation cannot therefore account for the larger aggregates from +/ +
than from T\T cells.
Figure 2 shows the initial pattern of disappearance of free cells (27-30-somite
stage). For the first 30min single T\T cells disappeared as fast as +/+ cells;
thereafter, the rate of decline of the free T\T cells was much reduced relative to
+ /+ cells. After 17 h of gyratory culture, less than 5% of the input cells in
both + / + and J / r cultures were free. The initial phase (the first 30min) of
cell aggregation was not inhibited by 0-5 /*g/ml of cycloheximide in either + / +
or T\T cultures, but the later phase was inhibited. Cycloheximide at this concentration inhibited the uptake of [3H]leucin in over 95 % of the cells.
The process of aggregate formation was followed, using cells from + / + and
TjT embryos at 27-30-somite stage. In + / + culture, the size of the aggregates
increased up to 7 h, then showed little change for the next 13 h. In TjT culture,
aggregation proceeded as for + / 4- cells for the first hour, then the difference
in size of the aggregates became progressively more distinct (Fig. 3), although
the aggregates rounded up at the same time in both types of culture.
EJ
Aggregation o/T/T cells
283
DISCUSSION
Cells isolated from + / + embryos formed progressively larger aggregates as
the embryos develop up to the 27-somite stage. With TjT cells, the aggregates
were smaller than those of + / + cells at all stages studied. The average diameter
of a single cell was similar for + / + and TjT cells. Over 83 % of the input cells
were recovered in the aggregates in both + / + and T\Tcultures. There was no
sign of preferential cell death of TjT cells during or after the formation of the
aggregates.
It is possible that the aggregation of T\Tcells reflects a general retardation of
development of T\T embryos. The cells from TjT forelimb-buds, however, form
smaller aggregates than those from + / + forelimb-buds, even though they are
morphologically similar and formed at the comparable stage of development.
Two possibilities exist concerning the adhesiveness of cells that form smaller
aggregates. One, proposed by Moscona (1961), assumes that aggregate size is
directly related to cell adhesiveness. The other hypothesis was proposed by
Ede & Agerbak (1968) who have several lines of evidence to show that size of
aggregate is inversely related to adhesiveness (Ede & Flint, 1975 a, b). There is
no direct evidence to indicate the relationship between adhesiveness and the
size of aggregates in our system; we can therefore only say that there seems
to be a difference between the adhesiveness of + / + and T\Tcells. Experiments
to discover if + / + and T\Tcells sort out are in progress.
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EDE,
(Received 5 January 1977, revised 16 March 1977)