Properties of Extra-Embryonic Ectoderm Isolated from Postimplantation Mouse Embryos

/. 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 ectoplacental 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 ectoderm 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 measurements 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. The results were expressed in the form of histograms of total absorbance, measured in arbitrary units. These histograms were then calibrated in multiples of the haploid DNA value (C) by comparison with and extrapolation from the liver controls, whose cells contain 2C, 4C, and 8 C values of DNA.

RESULTS Ectopic transfers The results of histological examination of the testes transfers are summarized in Table 1. The success rate of extra-embryonic ectoderm grafts of all ages was very high. All grafts identified were haemorrhagic and contained cells indis- tinguishable morphologically from trophoblast giant cells (Fig. 1). The extent of the haemorrhage and the size and number of giant cells varied but the grafts were similar to those produced by ectoplacental cones. Fewer embryonic grafts were identified and none produced obvious haemorrhage. Histologically, these grafts consisted of solid masses of relatively undifferentiated embryonic ectoderm-like cells (Fig. 2). Properties of mouse extra-embryonic ectoderm 187

50 fim

•»*

Fig. 1. Section of haemorrhage produced by 8^-day extra-embryonic ectoderm after 1 week under the testis capsule. Arrows indicate definite giant cells. Fig. 2. Section of graft produced by 7^-day embryonic ectoderm after 1 week under the testis capsule. Most cells appear similar to embryonic ectoderm. 188 J. ROSSANT AND L. OFER

Table 1. Tes'tis transfers of tissues from mouse egg cylinders, examined after 7 days

Type of tissue identified A ( Type of Age of No. No. Many tissue embryo No. haemorrhagic histologically trophoblast Undifferentiated transferred (days) transferred grafts identified giant cells diploid cells Extra-embryonic 51 8 5 5 5 ectoderm 61 5 5 5 5 — 71 11 10 10 10 81 7 7 7 7 Embryonic 51 10 0 2 2 ectoderm± 61 10 0 4 — 4 mesoderm 71 9 0 8 8 81 7 0 4 4 Ectoplacental 61 4 4 4 4 — cone 71 1 1 1 1

Table 2. In vitro culture of tissues isolated from mouse egg cylinders, examined after 7 days

Predominant cell types in explants Age of Type of tissue embryo No. No. alive Trophoblast Epithelial- Differentiating explanted (days) explanted after 7 days giant cells like cells muscle and nerve Extra-embryonic 51 29 25 25 ectoderm 61 32 27 27 71 17 16 16 81 12 11 11 Embryonic 51 18 3 3 ectoderm ± 61 42 13 10 3 mesoderm 71 17 17 4 13 81 12 11 11 Ectoplacental cone 51 8 3 3 61 30 28 28 71 5 5 5 81 2 2 2

In vitro culture The results of culture of embryonic and extra-embryonic fragments are summarized in Table 2 and confirm the results of ectopic transfers. Again the success rate of extra-embryonic ectoderm development was high and the cells resembled those produced by ectoplacental cones in both size and morphology after 1 week in culture. Embryonic ectoderm fragments often produced differentiating tissues but no morphologically giant cells were observed. After 1 week in culture, the cell number of the extra-embryonic fragments did Properties of mouse extra-embryonic ectoderm 189

Table 3. Mitotic index of tissues isolated from mouse egg cylinders after various times in culture

Mitotic index (no. metaphases/no. cells x 100) ± standard error, after 2 h colcemid treatment Type of tissue Age of embryo explanted (days) 0 day explants 1 day explants 2 day explants

Extra-embryonic 5* 151 ±30 2-4 ±1-2 0 ectoderm 16-8 ±1-4 3-4±l-5 0-4 ±0-2 20-9+11 3-4±0-l 21 ±01 10-1 ±3-2 l-6±0-3 0-9±0-6 Embryonic 7* 13-9 ±0-8 10-4 ±0-7 19-3 ±3-8 ectoderm 8* 11 0 + 0-4 9-5 + 1-3 8-9 + 0-6 Note: The mitotic index in each case is the average of at least three samples. The cell number in each sample ranged from 60 to 1700. not seem to have increased greatly, while the embryonic fragments were often very large. This suggested that little if any cell division had taken place following explanation of the extra-embryonic ectoderm fragments, and so the mitotic index of these explants was examined early in the culture period. The results are presented in Table 3. The mitotic index of the extra-embryonic ectoderm when isolated from the embryo is as high or higher than that of the embryonic ectoderm, but after only 24 h in culture it has dropped dramatically. After 2 days in culture the number of metaphases observed in the extra-embryonic ectoderm explants is extremely low and in the 5^-day explants, cell division has ceased entirely. The mitotic index remains high in the embryonic regions.

Microdensitometry Microdensitometry measurements confirm that cells with high DNA contents are produced in vitro by isolated extra-embryonic ectoderm from 5^- to 8^-day embryos. The embryonic ectoderm explants produced no giant cells and most cells had DNA values around 2C (Fig. 3). The peak was actually slightly below the 2C level but this may be an artifact since no correction was made for the obvious condensed nature of these nuclei (Goldstein & Bedi, 1974). It can also be seen that contamination with tetraploid feeder cells is minimal. By contrast, there are very few extra-embryonic ectoderm cells which fall into the diploid class after 1 week in culture, and some reach DNA values of 128 C or more (Fig. 4). The peak DNA value seems to be 8 C for all stages analysed but the percentage of cells falling into the 0-8 C range varies with age at ex- plantation. For 5^-day extra-embryonic ectoderm, 37 % of the cells fall into the 0-8 C range, while at 6^ days, this percentage rises to 57 %. At 1\ days, the percentage is 60 % and at 8^- days, it is 87 %. The larger proportion of cells with DNA values greater than 8 C from 5^-day extra-embryonic ectoderm may 13 EMB 39 190 J. ROSSANT AND L. OFER

51-day embryonic ectoderm (B) 61-day embryonic ectoderm 2C 4C 2C 4C 8C (A) 200 - 120r 180 - 100 - 160 = 80 - o o 140 2 60 120 5- 40 100

80 1 Ul_ 25 50 75 100 60 Total absorbance Total number of cells = 593 40 71 clay embryonic ectoderm 20 (Q 2C 4C 8C 0 100 0 25 50 75 100 Total absorbance 80 Total number of cells = 803

60 f 40

81 day embryonic ectoderm (D) 2C . 4C 25 50 75 100 70 I 1 Total absorbance Total number of cells = 358 60 Liver control 50 2(3 4C 8( 40 40

1 30 - n 30 o S 20 - 20 JO 1 10 0 25 50 75 100 25 50 Total absorbance Total absorbance Total number of cells = 216 Total number of cells = 536 Properties of mouse extra-embryonic ectoderm 191 be related to the fact noted previously, that they cease cell division in culture earlier than do explants from older embryos. Fig. 4E confirms that extra- embryonic ectoderm cells are diploid before culture.

DISCUSSION Extra-embryonic ectoderm isolated from the mouse embryo as late as 8^- days, when the chorion is well developed, can form cells with the morphological characteristics of trophoblast giant cells, both in ectopic sites and in vitro. Microdensitometry measurements have shown that cells containing more than 128 x the haploid DNA value can develop from extra-embryonic ectoderm in culture. The similarity between the properties of extra-embryonic ectoderm and ectoplacental cones at all stages up to 8^ days provides further support for the postulated common origin of these tissues from the trophectoderm of the blastocyst (Gardner & Papaioannou, 1975). Retention of the capacity to form giant cells by the isolated extra-embryonic ectoderm, which may never produce giant cells in the intact embryo, suggests that the mural giant cells and polar diploid trophoblast cells, which can first be distinguished at the late blastocyst stage, may not represent two distinct determined populations (Gardner & Rossant, 1976). There is no evidence that giant trophoblast cells can ever return to the diploid, mitotically active state (Zybina & Tikhomirova, 1963; Zybina & Mos'yan, 1967) but it is possible that the diploid trophoblast cells always retain the capacity to produce giant cells. It has previously been shown by many workers, notably Grobstein (1950), that ectoplacental cones isolated as late as 12 days of gestation will produce haemorrhage and giant cells when transferred to ectopic sites. However, such isolated cones contain many secondary giant cells as well as diploid trophoblast and it has not been proved conclusively that the diploid trophoblast cells can transform into giant cells. The present results provide an example of known diploid cells (Fig. 4E), of apparent trophectoderm origin (Gardner & Papaio- annou, 1975), retaining the capacity to form giant cells as late as 4 days after implantation. Later than 8^ days of gestation it becomes increasingly difficult to separate the diploid trophoblast from the overlying giant cells in the placenta, so that it is not yet known whether this property is retained by the diploid cells beyond this stage. It is not clear what maintains the extra-embryonic ectoderm in a diploid

Fig. 3. Histograms of DNA values of cells from explants of 5^ to 8^-day embryonic ectoderm after 1 week in culture. Figure 3E shows a control liver sample, with cells containing 2C, 4C and 8 C amounts of DNA. The labeled arrows on the other histograms mark the expected peak absorbance for a given C value, calculated from the liver controls. All slides were treated together, and the liver controls were similar in all; direct comparison between values of absorbance from different slides could therefore be made. All histograms represent the pooled data from two samples.

13-2 192 J. ROSSANT AND L. OFER (A) 51-day extra-embryonic ectoderm (B) 65-day extra-embryonic ectoderm o o o o oo o o o

HI, \ \ I 1 20 p 100r

15 - 80 cell s o o 10 - 60 "i 3 5 • 40 ol- Ulfl|Ul«,Mnnnmn 1 In n 1 1 20 0 500 1000 1500 2000 Total absorbance Total number of cells =127 500 1000 1500 2000 (C) 74-day extra-embryonic ectoderm Total absorbance Total number of cells = 583

(D) 8]- day extra-embryonic ectoderm OO O

250 500 750 1000 Total absorbance Total number of cells = 577

(E) 1\ day extra-embryonic ectoderm-non-cultured OO rj 20

15 375 Total absorbance L Total number of cells = 584 10

5 ln£L 25 50 75 100 Total absorbance Total number of cells = 152 Fig. 4. Histograms of DNA values of cells from explants of 5£ to 8i-day extra- embryonic ectoderm after 1 week in culture. Haploid DNA (C) values were calculated from the liver control readings (Fig. 3E). Fig. 4E shows the DNA values of cells from 7-^-day extra-embryonic ectoderm before culture. All histograms represent the pooled data from two samples. Properties of mouse extra-embryonic ectoderm 193 state in the intact embryo. At the blastocyst stage, contact with the ICM is thought to be necessary to maintain proliferation of the overlying trophectoderm cells, while cells away from the ICM endoreduplicate their DNA and transform into giant cells (Gardner, 1971, 1972; Barlow & Sherman, 1972; Gardner et ah 1973; Ansell & Snow, 1975). The fact that extra-embryonic ectoderm ceases cell division rapidly when separated from the rest of the embryo (Table 3) supports the suggestion that continued contact with ICM derivatives is necessary to promote postimplantation trophoblast proliferation (Gardner, 1975). This may be a specific inductive effect or the embryonic tissues may simply serve to maintain the organization and close cell contacts of the proliferating extra-embryonic ectoderm, since formation of giant cells by isolated extra-embryonic ectoderm is associated with disorganization of its normal structure as well as isolation from embryonic tissues.

We should like to thank Drs R. L. Gardner and C. F. Graham for useful discussion and Dr M. J. Evans and University College London for use of the microdensitometer. The work was supported by the Medical Research Council. J.R. is a Beit Memorial Junior Research Fellow.

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{Received 21 October 1976, revised 3 February 1977)