/. Embryol. exp. Morph. Vol. 39, pp. 183-194, 1977 183 Printed in Great Britain Properties of extra-embryonic ectoderm isolated from postimplantation mouse embryos By J. ROSSANT AND L. OFER1 From the Department of Zoology, Oxford SUMMARY Extra-embryonic ectoderm isolated from the mouse embryo as late as 8i days post coitum can form cells with the morphological characteristics of trophoblast giant cells both in ectopic sites and in vitro. This similarity to the properties of ectoplacental cone tissue provides further support for the postulated common origin of both tissues from the trophectoderm of the blastocyst. INTRODUCTION Embryologists have disagreed over whether the extra-embryonic ectoderm of the mouse egg cylinder arises from the trophectoderm (Jenkinson, 1900) or the inner cell mass (ICM) (Robinson, 1904) of the blastocyst. The extra- embryonic ectoderm lies between the ectoplacental cone and the embryonic ectoderm in the early egg cylinder. It later forms the ectoderm of the chorion which fuses with the ectoplacental cone to produce the trophoblastic layers of the placenta (Duval, 1891; Jenkinson, 1902). If the extra-embryonic ectoderm is derived from the trophectoderm of the blastocyst, this would suggest a unitary origin of all trophoblast tissues, since the trophectoderm is already known to give rise to the ectoplacental cone (Gardner, Papaioannou & Barton, 1973). Recent experiments tend to support this hypothesis. 'Reconstituted blastocyst' experiments revealed a trophectoderm contribution to the 'embryo plus membranes' fraction of later conceptuses (Gardner et al. 1973). Injection of rat ICMs into mouse blastocysts suggested that this contribution was to the extra- embryonic ectoderm, since no rat cells were ever found in the extra-embryonic ectoderm of resulting interspecific chimaeras, even where all the embryonic ectoderm was of rat origin (Gardner & Johnson, 1973, 1975). On the basis of this evidence, Gardner & Papaioannou (1975) suggested that the trophectoderm cells over the ICM of the blastocyst proliferate and push inwards to form the extra-embryonic ectoderm as well as outwards to form the ectoplacental cone (see figure 1, Gardner & Papaioannou, 1975). If this interpretation is correct, the distinction usually drawn between the ecto- placental cone and the extra-embryonic ectoderm at the origin of Reichert's membrane (Snell & Stevens, 1966, figure 12.8) is purely arbitrary and the two 1 Authors' address: Department of Zoology, South Parks Road, Oxford 0X1 3PS, U.K. 184 J. ROSSANT AND L. OFER tissues should have similar properties. Preliminary experiments suggest that this is so. 5^-day extra-embryonic ectoderm grafted under the testis capsule produced haemorrhagic grafts containing giant cells similar to those produced by grafted ectoplacental cones (Gardner & Papaioannou, 1975). Diwan & Stevens (1975) report similar results with 6-day extra-embryonic ectoderm. Extra-embryonic ectoderm isolated from embryos on the 6th and 7th days of pregnancy also formed cells morphologically similar to trophoblast giant cells when grown in vitro (Gardner & Ofer, unpublished results). The aim of the present experiments was to extend these preliminary studies to discover how late in development isolated extra-embryonic ectoderm retains the capacity to form giant cells. The isolated tissues were either transferred to ectopic sites, or cultured in vitro so that the cells could be readily harvested for mitotic counts and microdensitometry measurements. MATERIALS AND METHODS Recovery of embryos and separation of isolated germ layers Mice from random-bred Swiss PO stock (Pathology, Oxford) were used throughout this study. PB1 medium (Whittingham & Wales, 1969) containing 10 % foetal calf serum was used for recovery, storage, dissection and transfer of embryos. Embryos were dissected from the uteri of mice on the 6th, 7th, 8th and 9th day of pregnancy (5^-, 6^-, 1\-, 8^-day embryos). Both 5^- and 6^-day embryos consist of egg cylinders with no mesoderm formation (Snell & Stevens, 1966, figure 12.8). In 7^-day embryos, mesoderm is being formed by the primi- tive streak and the amniotic folds begin to separate the extra-embryonic ecto- derm from the embryonic shield (Snell & Stevens, 1966, figure 12.13). From this stage onwards the extra-embryonic ectoderm forms the ectoderm of the chorion. By the next day the amnion is complete and the chorion is flattened against the ectoplacenta, constricting the ectoplacental cavity. The allantois is also de- veloping but has not yet fused with the chorion (Snell & Stevens, 1966, figure 12.17). Somite formation has begun. After %\ days, clean separation of the chorionic ectoderm from the allantois and the ectoplacental cone is not readily achieved. Reichert's membrane was torn away from the embryos after they were cleared of uterine tissue and the embryos were cut by hand using glass micro- needles. At 5^ and 6^ days, embryos were divided into ectoplacental (very small and easily damaged at 5% days), extra-embryonic and embryonic regions. Since no obvious division between the ectoplacental cone and the extra-embryonic ectoderm was apparent, an arbitrary cut was made below the point of insertion of Reichert's membrane. At 1\ and 8-^- days, the same divisions were made, but an extra region including the amniotic folds was removed from the middle of the egg cylinder (the exocoelomic region) and discarded. The embryonic and extra-embryonic regions from each embryo were then Properties of mouse extra-embryonic ectoderm 185 placed in a solution of 2-5 % Pancreatin and 0-5 % trypsin in calcium, mag- nesium-free Tyrode's saline at 4 °C for 10-20 min. Incubation in this enzyme solution has been shown to facilitate separation of the germ layers in rat egg cylinders (Levak-Svajger, Svajger & Skreb, 1969). Separation of the germ layers in the present experiments was achieved by sucking the embryonic or extra- embryonic fragments up and down in flame-polished micropipettes of slightly narrower diameter than that of the fragments. At 5^ and 6^ days, enzymic treatment removed endoderm from the outside of the extra-embryonic ectoderm. At 1\ days, endoderm and extra-embryonic mesoderm came free from the chorionic ectoderm, and endoderm and some of the mesoderm were separated from the embryonic ectoderm. At 8^ days, endoderm and mesoderm were again removed from the chorionic ectoderm but no attempt at germ layer separation of the complex embryonic region was made. The results of this combined microsurgical and enzymic treatment was to produce clean fragments of extra-embryonic and embryonic ectoderm, with or without some attached mesoderm, from all stages. Ectoplacental cones were not subjected to enzyme treatment. Ectopic transfers Single embryonic ectoderm, extra-embryonic ectoderm and ectoplacental cone fragments were transferred beneath the testis capsule of male mice using a micropipette. At 8^ days, the fragments from a single embryo were cut into smaller pieces before transfer. After 7 days, the recipients were killed and their testes examined for the presence of haemorrhagic graft sites. All testes, whether or not showing macroscopic signs of graft survival, were fixed, embedded and sectioned at 7-8 /«n. The sections were stained with haemalum and eosin and scanned for the presence of graft derivatives. In vitro culture Embryonic ectoderm, extra-embryonic ectoderm and ectoplacental cone fragments prepared as above were cultured in separate wells of Falcon Micro- test II tissue plates at 37 °C. RPMI medium (Flow Labs)+ 10% foetal calf serum + 2 % glutamine, gassed with 5 % CO2, 90 % N2, 5 % O2, was used for culture and all fragments were grown on a feeder layer of Mitomycin C-treated mouse fibroblasts (Sto cells) at a concentration of 4 x 104 cells/well. A feeder cell layer was found to promote growth of the embryonic fragments and so was used for all explants to standardize culture conditions. The medium was changed every 2 days for 1 week. After this time, the cell morphology of the explants was recorded in the inverted phase microscope and some representative explants were prepared for microdensitometry. The mitotic activity of 5^- to 8^-day extra-embryonic ectoderm and 1\- and 8^-day embryonic ectoderm was also assessed. Explants were cultured in RPMI +1 /*g/ml colcemid for 2 h at 37 °C, either directly after dissection from the 186 J. ROSSANT AND L. OFER embryo or after 1 or 2 days in culture. The fragments were then fixed in acetic alcohol (1 part acetic acid/3 parts ethanol) and dissociated on to glass slides in 60 % acetic acid (Evans, Burtenshaw & Ford, 1972). The cells were mounted and stained in a toluidine blue/mountant mixture (Breckon & Evans, 1969) and the number of metaphases and interphase nuclei was counted. For smaller explants, total cell counts were made but for larger fragments the slides were scanned at intervals and all cells in each scan were counted. Microdensitometry Cell samples for microdensitometry were prepared by clearing away all feeder cells from the edges of the explants and then trypsinizing the embryo- derived cells from the plastic. The trypsinized cells were dried on to a clean glass slide and fixed in acetic alcohol for 1-8 h. After washing in absolute ethanol the slides were stored dry and dust-free. A mouse liver imprint was also placed on each slide and treated in the same way. The cell spreads were stained by the Feulgen technique for DNA (Pearse, 1972). Microdensitometry measure- ments were made using a Quantimet 720 system (Image Analysing Computers LW, Cambridge Instruments Ltd) with a Balzer K4 green interference filter, peak transmission between 550 and 560 nm. A densitometer module was used to digitize the optical density in steps of 0-02 absorbance units and sum these for each picture point. The numerical value given is Zd/32 = D and the absolute total integrated density is 32 x 0-02 x D. Control liver readings were made for each slide. In all samples every cell in a single scan was measured, but the whole of the sample was not always scanned.