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Cell Differentiation in Isolated Inner Cell Masses of Mouse Blastocysts in Vitro: Onset of Specific Gene Expression

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Cell Differentiation in Isolated Inner Cell Masses of Mouse Blastocysts in Vitro: Onset of Specific Gene Expression /. Embryol. exp. Morph. Vol. 53, pp. 367-379, 1979 357 Printed in Great Britain © Company of Biologists Limited 1979 Cell differentiation in isolated inner cell masses of mouse blastocysts in vitro: onset of specific gene expression By MARIE DZIADEK1 From the Department of Zoology, University of Oxford, U.K. SUMMARY Inner cell masses (ICMs) were isolated by immunosurgery from giant blastocysts formed by the aggregation of three morulae. A layer of endoderm cells formed on the outer surface of these primary ICMs in vitro. When this layer was removed by immunosurgery, a secondary endoderm layer formed. Alphafetoprotein (AFP) was used as a biochemical marker to characterize visceral endoderm formation in these cultured ICMs. The immunoperoxidase reaction on sections of ICMs cultured for intervals up to 120 h in vitro showed that some primary endoderm cells contained AFP, but these were always in the minority. The secondary endoderm layer, on the other hand, was composed of predominantly AFP-positive cells. It is concluded that the primary endoderm contains mainly parietal endoderm cells, while the secondary layer contains visceral endoderm cells. A model is proposed for the consecutive differentiation of parietal and visceral endoderm cell types from the ICM of mouse blasto- cysts. INTRODUCTION The mouse blastocyst on the fourth day of gestation consists of two cell types: an outer layer of trophectoderm cells and an inner cell mass (ICM). On the fifth day of gestation a morphologically distinct epithelial-like layer forms on the blastocoelic surface of the ICM. This layer of cells consistitutes what has been termed the primitive enoderm (Enders, 1971; Nadijcka & Hillman, 1974) and is thought to be the stem cell population which gives rise to the parietal and visceral endoderm cells in the later egg cylinder. When ICMs are isolated from the trophectoderm layer by either microsurgery (Rossant, 1975) or immunosurgery (Solter & Knowles, 1975; Strickland, Reich & Sherman, 1976; Pedersen, Spindle & Wiley, 1977), a complete outer layer of endoderm forms within 24 to 48 h of culture. This endoderm layer appears to be composed of a mixture of parietal and visceral endoderm cells, using morphological criteria (Solter & Knowles, 1975; Pedersen et al, 1977). Pedersen et al. (1977) have shown that a second layer of endoderm can 1 Author''s present address: Division of Biology, Kansas State University, Manhattan, Kansas 66506, U.S.A. 24-2 368 MARIE DZIADEK 'regenerate' when the first is removed from giant ICMs formed from aggrega- tions of between 4 and 12 morulae. This second layer was morphologically similar to the first. To characterize the type of endoderm cells formed on isolated ICMs it is necessary to have tissue-specific markers for both parietal and visceral endoderm cells. Plasminogen activator has been used as a marker for parietal endoderm cells in differentiating ICM cultures (Strickland, et al., 1976), but recent studies have shown that plasminogen activator is also secreted by ectoderm, mesoderm and visceral endoderm tissues from the mouse embryo and is, therefore, unsuit- able as a tissue-specific marker (Bode & Dziadek, 1979). Visceral endoderm cells, on the other hand, synthesize alphafetoprotein (AFP), a fetal serum protein, which can be used as a specific biochemical marker to characterize this cell type in vivo and in vitro in the absence of fetal hepatocytes which are the only other source of AFP in later embryos (Dziadek & Adamson, 1978). Parietal endoderm cells do not synthesize or accumulate AFP at any stage of develop- ment (Dziadek & Adamson, 1978). Visceral endoderm cells only fail to syn- thesize AFP when they are closely associated with extra-embryonic ectoderm tissue (Dziadek, 1978) and perhaps all trophectoderm-derived tissues, and under such situations visceral endoderm cells would not be recognized. ICMs can be isolated free of all trophectoderm cells from fully expanded blastocysts by immunosurgery (Solter & Knowles, 1975; Handyside & Barton, 1977; Hogan & Tilly, 1978 b). Hence AFP can be used as a reliable biochemical marker to characterize visceral endoderm formation in cultured ICMs. In the present study a comparison is made of AFP expression in the first endoderm layer to be formed on isolated ICMs and the second which 'regene- rates' when the first is removed. MATERIALS AND METHODS Giant blastocyst formation Embryos used in all experiments were the F2 progeny of Fx (CBA x C57BL) natural matings. Giant blastocysts were formed by the aggregation of three 8-cell-stage embryos to increase the size of the ICMs for easy experimental manipulation and immunohistochemistry. 8-cell-stage embryos were recovered from pregnant females on the morning of the third day of gestation (9.00-12.00 h, the day of the copulation plug being designated the first day of pregnancy). Oviducts with the upper third of the uterus were dissected into pre-warmed and pre-equilibrated Whitten's medium (Whitten, 1971). Embryos were most often located in the last loop of the oviduct before the oviduct-uterine junction, and were released by tearing the loop open with fine watchmaker's forceps under a Wild M5 dissecting microscope. In some mice, embryos had already entered the uterus, and so in each case the upper part of the uterus was also torn open. Embryos were transferred to fresh Cell differentiation in isolated ICMs 369 Whitten's medium. The zonae pellucidae were removed by placing embryos in pronase at 4 °C, and incubating for 10-15 min at 37 °C (0-5 % pronase dialyzed against phosphate buffered saline, PBS, Solution A of Dulbecco & Vogt, 1954) followed by gentle pipetting in Whitten's medium. Morulae were aggregated in groups of three in microdrops (2-5 fi\) of Whitten's medium under paraffin oil (Boots Pure Drug Co., U.K., specially selected to be non-toxic) on bacterio- logical grade plastic petri dishes (Sterilin, Richmond, Surrey, U.K.) and cultured at 37 °C in a sealed box which had been gassed with humidified 5 % CO2, 5 % O2, 90 % N2. Cultures were maintained for 48 h until fully expanded blastocysts were formed. This was found to be approximately 12 h later than when single morulae were cultured in the same way. Isolation of ICMs by immunosurgery The trophectoderm layer of blastocysts can be selectively killed using a two- step cytoxicity procedure which involves pre-incubation with anti-mouse anti- serum followed by exposure to complement (Solter & Knowles, 1975). The trophectoderm cells are connected by tight junctions which prevent antibody binding to the inner cells, and so the ICM is protected from complement- mediated lysis. (a) Preparation of anti-mouse antiserum Conceptuses together with placental tissue were dissected from C3H and F2 (CBA x C57BL) female mice on the 13th—15th days of gestation. All tissue was washed, minced and homogenized in PBS. After low-speed centrifugation for 5 min, the supernatant was collected and used as the material for injection. A female rabbit was injected intradermally at monthly intervals, the first injection with the embryonic extract plus Freund's complete adjuvant (50:50 v/v) and the two subsequent injections with the embryonic extract plus Freund's incomplete adjuvant (50:50 v/v). Blood was collected one week after the final injection. The serum was heated at 56 °C for 30 min to inactivate rabbit complement and was left unabsorbed since no specificity was required. (b) Immunosurgery procedure The anti-mouse antiserum was used at 1/10 dilution in Whitten's medium. Embryos were exposed to antiserum in a 0-5 ml volume for 30 min in a gassed incubator at 37 °C and subsequently washed three times in fresh Whitten's medium. Embryos were then exposed to 1/10 dilution of guinea-pig complement (Flow Labs, Ayrshire, Scotland) for 15 min and washed again in fresh Whitten's medium. ICMs were observed to round up after complement treatment. The lysed trophoblast layer was easily removed by pipetting the blastocysts through a fine-bore glass pipette, leaving a clean, smooth surface on the isolated ICMs. To test whether all trophectoderm cells had been removed, seven isolated ICMs were cultured on tissue-culture-grade petri dishes and observed over a 5-day 24-3 370 MARIE DZIADEK period for the outgrowth of trophoblast giant cells (Hogan & Tilly, 1978 a, b). No giant cells formed in these cultures, and it was therefore assumed that all trophectoderm cells were removed by the immunosurgery procedure. Culture oflCMs ICMs were cultured in microdrops (approximtely 5 jul) of a-medium, lacking nucleosides and deoxynucleosides, supplemented with 10% fetal calf serum (Stanners, Eliceiri & Green, 1971) under paraffin oil (Boots), gassed with humidified 5 % CO2 in air at 37 °C. Bacteriological grade plastic Petri dishes (Sterilin) were used to prevent adherence of ICMs, and hence maintain them in suspension culture. Cultures were maintained for up to five days, with medium being changed every two days. Analyses of AFP activity were made on each day of culture. In some cases the primitive endoderm layer was removed from ICMs by immunosurgery after 24-48 h in culture (Fig. 1). The procedure was identical to that described above for removing the trophectoderm layer. Alphafetoprotein analyses ICMs were analyzed for cellular localization of AFP at 24 h intervals by the immunoperoxidase technique on tissue sections (Dziadek & Adamson, 1978). ICMs were fixed by the Sainte-Marie technique (Sainte-Marie, 1962) using Engelhardt's modification (Engelhardt, Goussev, Shipova & Abelev, 1971). After fixation ICMs were stained in 1 % Eosin Y (Sigma) for 30 sec and then dehydrated. This allowed visualization of the ICMs during the embedding procedure and identification of tissue within wax sections. The preparation of the anti-AFP antiserum and the tests for its specificity have been described previously (Dziadek & Adamson, 1978). The histological preparation of tissue sections and the procedure for incubation in antisera and subsequent reaction with diaminobenzidine are also outlined in the report cited above.
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