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Sequential development and tissue organization in whole mouse embryos cultured from blastocyst to early somite stage.

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THE ANATOMICAL RECORD 197:317-329 (1980)
Sequent ial Development and Tissue 0rgan izat io n
in Whole Mouse Embryos Cultured From Blastocyst
to Early Somite Stage
BISHARAH L. LIBBUS AND YU-CHIH HSU
Labomtory of Mammalian Development and Oncogenesis, Department of
Pathobiology, School of Hygiene and Public Health, The Johns Hopkins
Uniuersity, Baltimore, Md. 21205 U S A .
ABSTRACT
The development of mouse embryos in culture from the implantation to the head-fold stage was sequentially examined. Our goal was to compare the
morphology of embryos grown in vitro to those developed in vivo, published in
standard texts, and to delineate the stages involved in the process of tissue differentiation and organization. Mouse blastocysts (stage 6) were collected a t 3.5 days
p.c. and cultured. Attachment of the blastocysts occurred on the second day of
culture (stage 8). Following the collapse of the blastocyst endoderm cells began to
migrate and to encircle the inner cell mass. At 2 days in culture the embryonic and
extra-embryonic ectoderm became distinguishable and the proamniotic cavity
appeared (stage 9).Egg cylinders began to project above the substrate a t 2.5 days in
culture (stage 10) and to progress through the stages observed in vivo. At 4 days a
posterior amniotic fold began to form (stage 11) and was followed a t 5 days by the
formation of the chorion, the appearance of mesoderm, exocoelom, and head fold
(stage 12). At 6 days in culture the embryo had differentiated longitudinally and
developed an allantois, blood islands, Reichert's membrane, head process, and
primitive streak. At 7 days somites as well as the neural fold and heart were
observed (stage 14) and were followed by further differentiation a t 8 days (stage
15). These observations indicate that apparently normal embryo development can
be maintained in vitro through the early stages of organogenesis, thus providing a
unique opportunity for investigating the regulation of early mammalian development.
The sequence of development of the mammalian embryo has been based primarily on the
study of excised or in situ-fixed embryos. Several monographs and papers have been published detailing the development and differentiation of the mouse fetus (Reinius, '65; Snell
and Stevens, '66; Rugh, '68; Theiler, '72). With
the recent advent of embryo culture techniques, a few workers briefly examined the development and morphology of cultured mouse
embryos (Hsu, '72, '73; Hsu et al., '74; Solter et
al., '74; Wiley and Pedersen, '77). The in vitrodeveloped embryo provides a unique opportunity for investigating mechanisms which regulate development and for examining the process of tissue differentiation during embryogenesis. Since, in our hands, embryos develop
in culture from the blastocyst to the 10-12 somite stage, the monitoring and detailed examination of changes involved in differentiation
0003-276x180/1973-0317$02.30
0 1980, ALAN R. LISS, INC.
and organogenesis can now be pursued. We
consequently became interested in determining the sequential appearance and organization of tissues during the early stages of attachment and growth of mouse embryos in culture, as well as in assessing the similarity of in
vitro-grown embryos to those developed in
utero. This report seeks to provide a systematic
and detailed description of the development of
mouse embryos in culture, between stages 6
and 15, and specifically of the process of cell
migration and organization of the germ layers.
As the data will amply show, development of
mouse embryos in culture, though proceeding
a t a slightly slower rate, is strikingly similar to
that observed in vivo.
Received October 1, 1 9 7 9 accepted December 13, 1979
317
318
B.L. LIBBUS AND Y.-C. HSU
MATERIALS AND METHODS
Random bred CF-1 mice (Charles River Co.,
Mass.) were given 5 i.u. pregnant mare serum
gonadotropin (PMSG) (Organon Inc., West
Orange, N.J.) and, 48 h later, 5 i.u. human
chorionic gonadotropin (hCG) (Ayerst Laboratory, N.Y.). At 3.5 days following mating,
female mice were sacrificed by cervical dislocation and the uteri removed. About 200 blastocysts from 20 animals were collected by flushing the uterine horns with lml of culture medium CMRL 1066 (Gibco, N.Y.). Blastocysts
were cultured, at the rate of 10 blastocysts per
35mm culture dish (Falcon, Ca.), in 2ml CMRL
1066 medium supplemented with 20% serum.
The serum used was fetal calf serum (Gibco,
N.Y.) for the first 3 days of culture, and human
placental cord serum for the remainder of the
culture period (Hsu, '79). Embryos were cultured a t 37°C in a humidified atmosphere of 5%
CO,. No antibiotics were added to the medium,
which was changed daily. The age of the embryos in culture was calculated starting a t the
time of initiation of culture. At daily intervals,
between 0 and 8 days of culture, embryos were
fixed in 3% glutaraldehyde, post-fixed in 1%
osmium, dehydrated in graded ethanol, and
embedded in plastic. After polymerization at
60°C for 3 days, 1 pm sections were cut and
stained with toluidine blue and photographed.
Between 3 and 10 embryos were examined a t
each stage. The developmental stage of the embryos was determined using the charts of
Witschi ('72) and Theiler ('72).
RESULTS
Mouse blastocysts collected a t 3.5 days p.c.
(stage 6) were still enclosed in the zona pellucida and consisted of a rim of single trophoblast cells surrounding the inner cell mass
(ICM) (Fig. 1). After 24 hours in culture the
majority of blastocysts hatched, and a n increase in the size of blastocyst and ICM was
observed (stage 7 , Fig. 2). Following the loss of
the zona pellucida mouse blastocysts attached
to the culture dish (stage 81, usually a t the
mural trophoblasts (Fig. 3). At that stage the
mural and polar trophoblastic cells became
morphologically distinguishable. Mural
trophoblast cells were characterized by large
nuclei and vacuolated cytoplasm, while polar
trophoblast cells had smaller nuclei that were
round and contained a prominent nucleolus.
Cells of the ICM typically had large nuclei that
exhibited a complex nucleolus, while the surrounding endoderm cells had smaller nuclei
and darker cytoplasm that was highly vacuolated (Fig. 4). These characteristics became
more distinct as the blastocyst attached more
firmly to the substratum, a process which was
accompanied by the collapse and flattening of
the blastocyst (Figs. 43). Attachment appeared
to accelerate growth and to initiate rapid
changes in the size and organization of the
blastocyst. The increase in the number of cells
in the different tissue layers was accompanied
by complete obliteration ofthe blastocoel and in
the accentuation of the staining characteristics
and vacuolization of endoderm cells (Fig. 5,6).
These changes were followed by the gathering
of the polar trophoblast cells over the ICM (Fig.
6).
Embryos a t 2 days in culture underwent a
process of tissue reorganization while maintaining a rapid rate of growth. Endoderm cells
migrated around and encircled the ICM. They
appeared to achieve this movement by either
penetrating through (Fig. 7) or, more fiequently, by migrating under the trophoblast
cells which receded before them (Figs. 8,9). The
ICM thus became surrounded by endoderm
cells except in a small area where ICM cells
maintained contact with the polar trophoblast,s
(Fig. 9-11). The ICM cells appeared to undergo
reorganization, becoming radially distributed
(Fig. 9) around a proamniotic cavity that became visible in midsaggital sections (Fig. 10).
Fig. 1. Mouse blastocyst isolated a t 3.5 days p.c. (stage 6)
Fig. 2. Blastocyst in culture for 1 day has shed its zona pellucida (stage 7).
Fig. 3. A t 2 days in culture (stage 8) blastocysts attach to the culture dish, usually a t the mural trophoblast
cells opposite the ICM.
Fig. 4. Following attachment blastocysts collapse and become flattened.
Fig. 5. Polar trophoblast cells are gathered over the ICM.
Fig. 6. Increase in the number of ICM cells and in the stainability of the endcderm cells is observed following
attachment. Bar is 20 p m . BC, blastocoel; End, Endoderm; GC,giant cell; ICM, inner cell mass; mTB, mural
trophoblast; pEnd, parietal endodem; pTB, polar trophoblast; vEnd, visceral endoderm; ZP, zona pellucida.
SEQUENTIAL DEVELOPMENT OF CULTURED MOUSE EMBRY0s
319
320
B.L. LIBBUS AND Y.-C. HSU
At 2.5 days in culture (stage 9, Fig. 11)mouse
embryos acquired a distinct proamniotic cavity, began to grow and project above the substratum, and became surrounded by a layer of
endoderm cells which connected with the ectoplacental cone on both sides of the egg cylinder
(Fig. 11).The embryos continued to develop so
that a t 3 days in culture (Figs. 12,131,while the
ectoplacental cone appeared to grow slowly, the
egg cylinder constituted a progressively larger
portion of the embryo (stage 10, Fig. 13). The
embryonic and extra-embryonic ectoderm
layers became increasingly more delineated
and a line of demarcation became visible (Fig.
13-17). At 4 days in culture squamous and
cuboidal endoderm cells had differentiated and
a posterior amniotic fold could be observed
(stage 11, Fig. 14). Mesoderm cells were first
observed in embryos a t 5 days in culture (stage
12, Fig. 18), a t which time the embryonic ectoderm had differentiated into a recognizable
head process and primitive streak (stage 13,
Fig. 19). The posterior and anterior amniotic
folds came in contact, giving rise to the chorion,
the mesoderm became more developed, and a n
exocoelom was formed (Fig. 19). The ectoplacental cone had grown appreciably and exhibited an ectoplacental cavity (Fig. 19). A
Reichert's membrane formed covering the
placenta (Fig. 18).
Embryos began to loose their attachment to
the culture dish after 6 days in culture. At stage
13 (Fig. 19,20)cultured mouse embryos exhibited three distinct compartments. The ectoplacental cavity, the exocoelom, and the amniotic
cavity were separated by the chorion and the
amnion, respectively (Fig. 20). Blood islands
formed in the yolk sac splanchnopleure, the
chorion regressed toward the ectoplacental
cone, which in turn was surrounded by the
membrane of Reichert, and the allantois became prominent (Fig. 20). The embryo proper
consisted of ectoderm, head process, and primitive streak regions (Fig. 21). Embryos continued to develop in size and their convex shape
became more relaxed. At 6.5 days (stage 14,
Fig. 22) blood islands were common, the chorion was incorporated within the ectoplacenta,
and the sphere of the yolk sac containing the
embryo was detached and free-floating in culture. The embryo exhibited a primitive streak
with an attached allantois on one end, and a
neural fold, heart mesoderm, and foregut on the
other (Fig. 22,23). The heart mesoderm began
to be organized into endocardium and myocardium. The most striking change observed in
embryos cultured for 7 days was the progressive differentiation of the somites and the head
process. In embryos a t that age, 7-8 somites
developed (Fig. 24, 25). The heart consisted of
two separate chambers, the endocardium and
the ectomycocardium, and was beating regularly. The circulatory system became well developed and blood vessels could be seen (Fig.
25). The allantois became more developed and
elongated, stretching toward the yolk sac
splanchnopleure opposite the embryonic region
(Fig. 24). These changes were followed by further differentiation a t 8 days, a t which age the
somites numbered between 10 and 12 (Fig. 26).
The hind gut became apparent, heart beat became active and regular, and the circulatory
system was further developed as vitelline arteries became evident (Fig. 26). The neural fold
became differentiated into the mesencephalon
and two rhombomeres (Fig. 26). Between 40
and 50%of the culture blastocysts progressed to
stage 15 after 8 days in culture.
DISCUSSION
Growth and differentiation of the mammalian embryo involves complex and intricate
processes of cellular commitment and interaction. Culture conditions may also conceivably
introduce some deviation from normal development. However, comparison of mouse embryos grown in vitro (Fig. 3-26) with corresponding in vivo stages (Snell and Stevens, '66;
Rugh, '68; Theiler, '72) indicates close similarity and essentially indistinguishable developmental sequences. The differentiation of somites, heart, neural fold, blood islands, and
Fig. 7. Cellular movement and reorganization in mouse embryos cultured for 2 days (stage 8). Endoderm cells penetrate
the trophoblast cells and migrate to encircle the ICM.
Fig. 8. More commonly, endoderm cells migrate under the trophoblast cells and surround the ICM.
Fig. 9. Movement of the endodem cells appears to be faster a t one end, allowing the ICM to maintain contact with
trophoblast cells. ICM cells appear to be radially arranged.
Fig. 10. A proamniotic cavity is first observed in mouse embryos cultured for 2 days. Bar is 20 pm. eEct, embryonic
ectodem; End, endoderm; Ep, epiblast; mTB, mural trophoblast; PAC, proamniotic cavity; pTB, polar trophoblast; xEct,
extra-embryonic ectoderm.
SEQUENTIAL DEVELOPMENT OF CULTURED MOUSE EMBRYOS
321
322
B.L. LIBBUS AND Y.-C. HSU
Fig. 11. Mouse embryos cultured for 2.5 days (stage 9) form egg cylinders that begin to project above the
substratum. The endoderm-covered ectoderm tissue is differentiated into embryonic and extraembryonicregions.
Bar is 20 pm.
Fig. 12. Embryo cultured for 3 days exhibits a well developed embryonic ectoderm layer. Bar is 20 pm.
Fig. 13. At 4 days in culture endoderm has differentiated into squamous and cubaidal cells, and a line of
demarcation is visible between the embryonic and extraembryonic ectoderm (arrow). Bar is 20 km.
Fig. 14. Embryo cultured for 4 days (stage 11) has developed a posterior amniotic fold. Embryonic ectoderm
exhibits progressive differentiation. Bar is 50 pm.
Fig. 15. Embryo cultured for 2 days showing cellular discontinuity between embryonic and extra-embryonic
ectoderm (arrows). Bar is 50 pm.
Fig. 16. This line of demarcation is apparent in embryos cultured for 4 days (arrows). Bar is 50 Km.
Fig. 17. Detail of embryo culturedfor 4days showing distinctdivision between the twoectodermregions. Bar is
50 pm. eEct, embryonic ectoderm; End, endoderm; EPC, ectoplacental cone; PAC, proamniotic cavity; pAF,
posterior amniotic fold; xEct, extraembryonic ectoderm.
SEQUENTIAL DEVELOPMENT OF CULTURED MOUSE EMBRYOS
323
324
B.L. LIBBUS AND Y.-C. HSU
Fig. 18. Embryo cultured for 5 days continue to develop, exhibiting a Reichert's membrant (stage 12) and mesoderm
tissue. Bar is 100 pm.
Fig. 19. A more advanced embryo, also cultured for 5 days, exhibits a head process, chorion, amniotic cavity, and
exocoelom. Bar is 100 pm. AC, amniotic cavity; Ch, chorion; EC, ectoplacental cavity; eEct, embryonic ectoderm; E X ,
ectoplacental cone; HP, head process; Mes, mesoderm; PAC, proamniotic cavity; pAF, posterior amniotic fold; P1, placenta;
RM, Reichert's membrane; XC, exocoelom; xEct, extra-embryonic ectoderm.
vessels indicates that early organogenesis in blastocyst (Fig. 3-6) and the subsequent migracultured mouse embryos is maintained under tion of endodermal cells to a dorsal position
the present culture conditions. The ultrastruc- while the trophoblast cells recede before the
ture of mouse embryos which developed in cul- advancing endoderm (Fig. 7-10). The net effect
ture from the blastocyst to the egg cylinder of such movement is that while the attachment
stage was shown to be similar to those devel- site of the blastocyst appears to be similar in
oped in utero (Solter, et al., '74). The apparently vivo and in vitro, the egg cylinder in culture has
normal development through the early or- rotated 180"in comparison to that in vivo (Fig.
ganogenesis stages supports the use of cultured 4, 11) (Reinius, '65; Snell and Stevens, '66;
embryos as a model system for investigating Rugh, '68, Theiler, '72). Following this maneuthe regulation of embryonic development and ver, an endoderm-covered egg cylinder contindifferentiation.
ues to grow and differentiate normally (Fig.
Since the blastocyst attaches a t the mural 14-26). It is of interest to note the clear line of
trophoblast end, growth of the embryo in cul- demarcation between the embryonic and exture requires rearrangement of its topography tra-embryonic ectoderm (Fig. 13-17), which
and germ layers. This is apparently accom- supports previous observations that these two
plished by the collapse and flattening of the tissues have different origins. It was shown
SEQUENTIAL DEVELOPMENT OF CULTURED MOUSE EMBRYOS
325
326
B.L. LIBBUS AND Y.-C. HSU
SEQUENTIAL DEVELOPMENT OF CULTURED MOUSE EMBRYOS
327
328
B.L. LIBBUS AND Y.-C. HSU
SEQUENTIAL DEVELOPMENT OF CULTURED MOUSE EMBRYOS
that the extraembryonic ectoderm and the ectoplacental' cone commonly originate from the
trophectoderm (Gardner, et al., '73; Gardner
and Papaioannou, '75; Rossant and Ofer, '77).
On the basis of the observed relative position of
the embryonic and extra-embryonic ectoderm,
it appears likely that the latter tissue originates from the polar trophoblasts which invade
the proamniotic cavity (Fig. 11-17).
A recent report by Wiley and Pedersen ('77)
described the development of mouse blastocysts
into stage 13 egg cylinders after 8 days in culture. This constituted a lag of 4.5 days behind
the comparable in vivo stage, and may have
been partially due to the fact that the medium
was not changed for the duration of the culture
period. In our hands cultured mouse embryos
reached the same stage after 5 days in culture
(Fig. 19). Use of human placental cord serum
and changing the culture medium daily (Hsu,
'79) have contributed significantly t o maintaining progressive differentiation of embryos in
vitro. Additional improvement of the culture
conditions will likely continue t o support further differentiation of embryos in culture and
narrow the differences between in vivo and in
vitro development.
LITERATURE CITED
Gardner, R.L., and V.E. Papaioannou (1975) Differentiation
of the trophectoderm and inner cell mass. In: The Early
Development of Mammals, M. Balls and A.R. Wild, eds.
Cambridge University Press, Cambridge, pp. 107-132.
329
Gardner, R.L., V.E. Papaioannou, and S.C. Barton (1973)
Origin of the ectoplacental cone and secondary giant cells
in blastocysts reconstituted from isolated trophoblast and
inner cell mass. J. Embryol. exp. Morphol., 3Ot561-571.
Hsu, Y.C. (1972) Differentiation in uitro of mouse embryos
beyond the implantation stage. Nature, 239~22CL202.
Hsu, Y.C. (1973)Differentiation in uitro ofmouse embryos to
the stage of early somite. Dev. Biol.,33:403-411.
Hsu, Y.C. (1979) In uitro development of individually cultured whole mouse embryos from blastocyst to early somite stage. Dev. Biol., 68t453-461.
Hsu, Y.C., J. Baskar, L.C. Stevens, and J.E. Rash (1974)
Development in uitro of mouse embryos from the two-cell
egg stage to the early somite stage. J. Embryol. exp. Morphol., 31:235245.
Reinius, S. (1965)Morphology of the mouse embryo, from the
time of implantation to mesoderm formation. Z.
Zellforsch., 68r7 11-723.
Rossant, J., and L. Ofer (1977)Properties of extraembryonic
ectoderm isolated from postimplantation mouse embryos.
J. Embryol. exp. Morphol., 39t183-194.
Rugh, R. (1968)The Mouse: Its Reproduction and Development. Burgess, Minneapolis.
Snell, G.D., and L.C. Stevens (1966)Early embryology. In:
Biology of the Laboratory Mouse. E.L. Green, ed. McGraw
Hill, New York, pp. 2 0 s 2 4 5 .
Solter, D., N. Biczyoko, M. Pienkowski, and H. Koprowski
(1974) Ultrastructure of mouse egg cylinders developed in
uitro. Anat. Rec., 180:263-279.
Theiler, K. (1972) The House Mouse. Springer-Verlag, New
York.
Wiley, L.M., and R.A. Pedersen (1977)Morphology of mouse
egg cylinder development in uitro: A light and electron
microscopic study. J. Exp. Zool., 200:30%402.
Witschi, E. (1972) Characterization of developmental stages,
11. Rat. In: Biology Data Book. P.L. Altman and D.S. Dittmer, eds. Fed. Am. Soc. Exp. Biol., Washington, D.C., Vol.
I, pp. 17S180.
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