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/ . Embryo/, exp. Morph. Vol. 53, pp. 335-344, 1979
Printed in Great Britain © Company of Biologists Limited 1979
335
Molecular differentiation of inside cells
and inner cell masses isolated from the
preimplantation mouse embryo
By M.H.JOHNSON1
From the Department of Anatomy, University of Cambridge
SUMMARY
Clusters of inside cells (ICs) were isolated immunosurgically from morulae and early
cavitating blastocysts, and cultured for varying periods in vitro. The ICs responded to their
changed position by transformation to a blastocyst-like vesicle and also showed a correlated
synthesis of trophectodermal-marker polypeptides. Both changes were inhibited bya-amanitin,
indicating that transcriptional events are involved in this response to changed position. Inner
cell masses (ICMs), isolated immunosurgically from expanded blastocysts, did not undergo a
morphological transformation to a blastocyst-like structure and did not show a corresponding
and complete change in synthesis of trophectodermal-marker polypeptides. Elements of the
change in polypeptide activity were, however, present, suggesting an ineffective response to
position by the ICMs.
INTRODUCTION
Each blastomere of the eight-cell mouse embryo evidently retains developmental totipotency (Kelly, 1977). At some point shortly thereafter, cells embark
on a restricted course of development in response to positional cues (Johnson,
1979). It is proposed that inside cells (ICs) at the morula stage develop into the
inner cell mass (ICM) of the blastocyst and outside cells (OCs) into the trophectoderm (Tarkowski & Wroblewska, 1967; Hillman, Sherman & Graham, 1972).
The time at which inside and outside positions are first recognized by embryonic
cells has been investigated by use of a set of intrinsic cell markers. The two
tissues of the 3^-day blastocyst may be differentiated from each other by their
synthesis of tissue marker polypeptides (Van Blerkom, Barton & Johnson, 1976).
When whole embryos at earlier developmental stages are analysed, they are also
found to synthesize some of these marker polypeptides, and, moreover, there
appears to be a relationship between the position of the cells within the morula or
early blastocyst and the type of polypeptides that they synthesize (Handyside &
Johnson, 1978). Thus, inside cells do not synthesize the trophectodermal-tissue
polypeptides but do synthesize lCM-tissue polypeptides (Handyside & Johnson,
1978). It seems likely from these results that cells recognize position, as reflected
by their molecular differentiation, at least as early as the morula stage.
1
Author's address: Department of Anatomy, Downing Street, Cambridge. U.K.
336
M. H. JOHNSON
Whilst populations of inside and outside cells taken from the morula and
early blastocyst may be differentiated from each other, their development along
a restricted lineage is not irreversible since, on their isolation and culture in vitro,
clusters of either inside or outside cells can give rise to blastocysts (Johnson,
Handyside & Braude, 1977; Handyside, 1978 a, b). Furthermore, blastocysts
derived from aggregates of ICs will implant in utero to yield both normal egg
cylinders and placental tissues (Handyside, 19786). Final commitment to ICM
formation at least does not appear to occur until late in blastocyst expansion
(Johnson et al. 1977; Handyside, 1978a, b; Surani, Torchiana & Barton, 1978;
Spindle, 1978; Hogan & Tilly, 1978), after which time ICMs do not contribute
to the trophectoderm lineage either in vivo (Rossant, 1975; Handyside, 19786)
or in short-term cultures in vitro (Handyside, 1978 a, b).
The spatially defined patterns of polypeptide synthesis which occur in
response to positional cues within the morula could be regulated either via
transcriptional control through selective gene expression or via post-transcriptional control through, for example, selection of cytoplasmic mRNA.
Indeed a transition with time from post-transcriptional to transcriptional control
could provide a molecular explanation for the apparent period of 'reversible'
differentiation prior to commitment. The experiments described in this paper
have attempted to discriminate between these alternatives.
MATERIALS AND METHODS
1. Embryos
Outbred 4- to 6-week-old CFLP mice (Anglia Laboratory Animals) were
superovulated, mated and embryos recovered at 84, 96 or 120 h post-HCG.
Dead or retarded embryos at each time were discarded; 84h embryos were
separated into morulae and early blastocysts. Zonae were removed by a 10 sec
exposure to acid tyrode's solution, and the embryos left for 30-60 min in phosphate-buffered medium 1 (PB1) before use.
2. Immunosurgery and culture
Immunosurgical recovery of clusters of inside cells (ICs) from morulae and
early cavitating blastocysts (84 h post-HCG) and of ICMs from fully expanded
96 or 120 h blastocysts was carried out by procedures shown by several techniques to yield cell clusters uncontaminated by outer cells, as described in detail
elsewhere (Handyside, 1978 a; Handyside & Barton, 1977). ICs or ICMs were
cultured in RPM1 + 1 0 % foetal calf serum supplemented with glutamine in an
atmosphere of 5 % CO2 in air. Some embryos were cultured for varying periods
of time in these conditions prior to immunosurgery as described in the text. In
some experiments, a-amanitin (11 /^g/ml, Boehringer Mannheim) was included
both in the medium in which the immunosurgery was performed and in the
medium for culture and labelling of embryos (Braude, 1979a).
Differentiation of cells isolated from mouse embryo
337
3. Labelling of embryos and electrophoretic separation of polypeptides
Embryos, or immunosurgically isolated parts thereof, were placed in 0-1 ml
of a protein-free medium 16 (Whittingham, 1971) containing 10 [A of [35S]methionine (~ 1000 Ci/mmole, Radiochemical Centre, Amersham) for 4 h as
described previously (Handyside & Johnson, 1978). Labelled embryos were
harvested into lysis buffer, frozen and thawed 3 x, and applied to cylindrical
gels for isoelectric focusing. The gels were then laid on gradient SDS polyacrylamide slab gels for electrophoresis according to molecular size (O'Farrell,
1975; as described in detail in Handyside & Johnson, 1978). Gels were fixed,
impregnated with PPO (Laskey & Mills, 1975), dried, and exposed to preflashed
Fuji Rx film or Kodak RP45 film (Bonner & Laskey, 1974). Films were coded
and analysed 'blind' as described in detail in Handyside & Johnson (1978) and
Braude (1979 a). Six trophectodermal-marker polypeptides designated 2, 3, 4, 6,
13 and 16 and described previously (Handyside & Johnson, 1978) were used as
markers of trophectodermal differentiation (see also legend to Fig. 1).
RESULTS
Inside cells (TCs) isolated from late morulae and early cavitating blastocysts
(84 h post-HCG) and cultured for 0, 8,16 or 24 h, showed signs of fluid accumulation in individual cells by 16 h and had formed blastocyst-like vesicles within
24 h, thus confirming previous observations (Handyside, 1978#). The patterns
of incorporation of [35S]methionine into the six trophectodermal-marker
polypeptides indicated in Fig. 1 are summarized in Fig. 2 A, B. For comparison,
Fig. 2C includes a summary of the patterns obtained for intact embryos 84 and
96 h post-HCG. It will be observed that after 24 h, followed by 4 h in label, the
ICs (84/24/4 in Fig. 2 A, B) show the same profile as intact blastocysts (96/4 in
Fig. 2C) and this correlated with their blastocyst-like morphology. Intact
embryos recovered at 84 h post-HCG were also cultured for 24 h, subjected to
immunosurgey and the ICMs labelled directly for 4 h. The polypeptide pattern
obtained is summarized in Fig. 2D. Further groups of embryos were left in vivo
until 108 h post-HCG, and after recovery their ICMs were immunosurgically
isolated and labelled directly for 4 h (Fig. 2D). The ICMs from intact embryos,
in contrast to those cultured in isolation, do not acquire or increase their
capacity to synthesize trophectoderm-marker polypeptides.
Blastocysts recovered from the uteri at 96 h post-HCG were subjected to
immunosurgery and the ICMs recovered were placed in culture for 0, 8, 16, 24
or 36 h prior to 4 h of labelling. At no time during the incubation did the cells of
the isolated ICMs show evidence of fluid accumulation, and blastocyst-like
vesicles did not form, thus confirming the results of Handyside (1978 a). The
polypeptide synthetic profiles of the cultured ICMs are summarized in Fig. 3.
Some trophectodermal-marker polypeptides show indications of increased
synthesis; however, none is as uniformly pronounced as in cultured ICs and
synthesis of all is greatly retarded over that observed for ICs.
338
M. H. JOHNSON
la)
SDS
10
pH7.0
IEF
pH4.5
Fig. 1. Fluorograph of [32S]methionine-labelled polypeptides synthesised by ICs
derived from mouse embryos (84 h post-HCG) and separated by two-dimensional
electrophoresis. Horizontal separation is by iso-electric focusing over the pH range
indicated. Vertical separation is by SDS gradient acrylamide electrophoresis giving
separation by molecular weight (as indicated by 1, 5 and 10 x 104 M.W.). The
position of the polypeptide markers for trophectoderm are indicated and numbered
2, 3, 4, 6, 13 and 16 (Van Blerkom et al. 1976; Handyside & Johnson, 1978).
(b) similar to (a) but after labelling performed after 24 h culture of the ICs.
Differentiation of cells isolated from mouse embryo
(A)
(C)
2 3 4 6 13 16
2 3 4 6 13 16
(84/0/4)
(84/4)
(intact
(84/8/4)
(96/4)
(intact)
(84/16/4)
(D)
0
(84/24/4)
(B)
3 4
6 13 16
(84+-24/0/4)
2 3 4 6 13 16
(108/0/4
(84/0/4)
(84/24/4)
Fig. 2. Diagram to indicate relative synthesis under different conditions of the six
tissue-specific polypeptides (2, 3, 4, 6, 13, 16) indicated in Fig. 1. Each small block
represents analysis of 1 gel for 1 polypeptide. • = no detectable synthesis;
[D = relatively weak synthesis compared to 96 h post-HCG blastocyst; • = comparable synthesis to that observed in 96 h post-HCG blastocyst; U = not scorable.
Panel A represents data from ICs isolated from early cavitating blastocysts (84 h
post-HCG), cultured in vitro for 0-24 h and labelled over a 4 h period. The numbers
at left represent in hours (age post-HCG/culture period/labelling period). Panel B
is the same for ICs isolated from morulae (84 h post-HCG). Numbers at left as for A.
Panel C represents data for intact early blastocysts (84 h post-HCG) or expanded
blastocysts (96 h post-HCG) after incubation in label for 4 h. Numbers at left
represent in hours (age post-HCG/labelling period). Panel D represents data for
ICMs derived from early blastocysts (84 h post-HCG) which were cultured intact
for 24 h either in vitro or in vivo (108 h at recovery), immunosurgically dissected,
and then the ICMs incubated directly in label for 4 h. Numbers at left as for A.
Number of gels analysed = 28 for A, 6 for B, 12 for C, 8 for D.
339
340
M. H. JOHNSON
»
(96/0/4)
(96/8/4)
(96/16/4)
3
4
6
13 16
_
T =ir
1
(96/24/4)
-
--
(96/36/4)
Fig. 3. Diagram, details as for Fig. 2, summarizing data for ICMs isolated from
blastocysts (96 h post-HCG) and incubated for 0-36 h in culture followed by 4 h in
label. Numbers at left as for Fig. 2 A. Number of gels analysed = 19.
Intact embryos (84 h post-HCG) and ICs derived immunosurgically from
them were also cultured for either 8 or 24 h in 11 /*g/ml a-amanitin. The results
of this culture are summarized inTable 1. Prolonged culture in a-amanitin resulted
in death of ICs, although ICMs in intact embryos were not prevented from
forming nor were they killed, as has also been reported by Braude (19796).
Inspection of cultures revealed that death of the ICs occurred between 12 and
14 h after isolation of the ICs in the presence of a-amanitin, that is prior to the
overt morphological transition to the blastocyst in control cultures. The polypeptide synthetic profiles of ICs incubated for 12 h in the presence of a-amanitin,
the last four of which were in the presence of labelled methionine, are shown in
Fig. 4.
Differentiation of cells isolated from mouse embryos
3
4
6
13
341
16
(84/0/4)
•
(84/8/4)
(control)
(84/8/4)
(+a--amanitin)
Fig. 4. Diagram, details as for Fig. 2, comparing data for ICs isolated from early
cavitating blastocysts (84 h post-HCG) and labelled directly (0 + 4), or after
incubation in vitro for 8 h in the absence or presence of a-amanitin (11 /*g/ml).
Numbers at left as for Fig. 2 A. Number of gels analysed = 12.
Table 1. Effect of incubation in 11 /.ig/ml ot-amanitin on development in.
vitro of intact 84 h post-HCG embryos, or of the ICs derived therefrom
Number c)f embryos showing indicated morphology
at the end of incubation
Time (h) and
nature of treatment
Morulae
Morulae with Blastocysts
fluid accumul- with live
ating cells
ICMs*
Dead
485
90
2
3
0
0
3
20
350
1
o o
a-Amanitin
Untreated!
24 h
a-Amanitin
Untreated!
o o
ICs (84 h post-HCG)
0
46
1
0
0
0
64
61
0
0
0
0
10
0
101
61
0
0
Intact embryos (84 h post-HCG)
ou
a-Amanitin
Untreated!
24 h
a-Amanitin
Untreated!
* As assessed both by direct observation and immunosurgical isolation,
! All controls done in parallel with test experiments.
342
M. H. JOHNSON
DISCUSSION
The ICs isolated from morulae and early blastocysts (84 h post-HCG) and
cultured in vitro, showed a change in their polypeptide synthetic profile that
corresponded temporally to their change in morphology. The acquisition of
marker polypeptides occurred in the same sequence as that reported for intact
differentiating morulae but with a lag of approximately 12-18 h on absolute
embryonic time (Handyside & Johnson, 1978). The observation that ICs left
within trophectoderm, in vivo or in vitro, over the same time period did not
undergo these changes in synthetic profile (Fig. 2D) suggests that this response
was abnormal and was elicited by the changed environment of the ICs.
The sensitivity to a-amanitin of ICs isolated in vitro compared with those
retained within trophectoderm suggests that transcriptional processes are
involved in a complete response to altered position. It is not clear, however, that
the very earliest molecular changes were totally blocked (Fig. 4). Those species of
polypeptide (2, 3, 6, 16), which showed a partial resistance to a-amanitin, are
identical to those observed in the incomplete synthetic response of isolated
ICMs (Fig. 3), and are also seen weakly in some fluorographs of freshly isolated
ICMs or ICs indicating either their synthesis at a low level or very rapid activation of their synthesis during the 4 h labelling period in response to positional
change. These considerations make it impossible to exclude a post-transcriptional control mechanism for some of the earlier events of the IC transformation
to a blastocyst. However, both these results and those using a-aminatin on intact
blastocysts (Braude, 19796), make it highly unlikely that post-transcriptional
controls play the sole and major role in controlling blastocyst formation
(Johnson, 1979). Perhaps a clearer resolution of this problem will come from
analysis of the in vitro translates of mRNA isolated from ICs at various times
after their immunosurgical recovery (Braude & Pelham, 1979).
In contrast to ICs, ICMs isolated from fully expanded blastocysts (96 h
post-HCG) did not acquire the complete set of trophectodermal-marker
polypeptides over the same time course (compare Fig. 2 A with Fig. 3). Neither
did they show evidence of a morphological transformation to a blastocyst.
However, elements of molecular differentiation in a trophectodermal direction
were detected, since synthesis of the earliest trophectodermal-marker polypeptides seen in the ICs was also seen in some ICMs, although less evident and
later in time of appearance. The ICMs thus do appear to respond to their
altered position by developing trophectoderm-marker polypeptides, but do so
more slowly and less completely than the ICs. Whilst synthesis of all the trophectodermal-marker polypeptides appears to be diagnostic for mature trophectoderm, synthesis of only some is not in itself an adequate criterion for the
diagnosis of trophectodermal cells. The less complete molecular response of the
ICM to positional change could be interpreted as an attempt to unscramble and
reverse differentiation by some or all of the cells in the ICMs. ICMs cultured
Differentiation of cells isolated from mouse embryo
343
in vitro for prolonged periods show only limited and probably abnormal
differentiation (Hogan & Tilly, 1978) and it has not been established that 24-36 h
culture in vitro is compatible with normal ICM function on relocation within a
trophectodermal vesicle. It is possible that cultured ICMs are abnormal because
of their incomplete or confused molecular responses to positional change in
these early stages of culture. This result is consistent with models for commitment which do not invoke a special, quantal molecular mechanism but rather a
progressive accumulation of differentiative changes (Johnson et al. 1977;
Johnson, 1979).
The author wishes to thank Jo Close and Gin Flach for their technical help, Drs P. Braude,
H. Pratt and A. Handyside for their criticism of the manuscript and Raith Overhill and Chris
Burton for their help in preparing figures. The work was supported by grants from the
Medical Research Council and the Ford Foundation to the author.
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(Received 2 May 1979, revised 5 June 1979)