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{"project":"2_test","denotations":[{"id":"16628246-10611232-85799756","span":{"begin":897,"end":899},"obj":"10611232"},{"id":"T19776","span":{"begin":897,"end":899},"obj":"10611232"}],"text":"Trophectoderm Failure in Atrxnull Embryos\nWhole-mount observation of 8.5 dpc embryos revealed that, in contrast to the basically normal although delayed morphology of the embryo itself, the extraembryonic tissues of Atrxnull conceptuses appeared highly disorganised. When embryos were removed from deciduas, the surrounding trophectoderm layer appeared dramatically reduced in Atrxnull embryos relative to wild-type littermates, and the underlying ectoplacental cone appeared reduced and abnormally shaped (Figure 7A). Vacated deciduas surrounding 8.5 dpc wild-type and Atrxnull embryos were bisected and analysed by WMISH for expression of placental lactogen-1 (Pl-1), a marker of terminally differentiated TGCs. The number of Pl-1-expressing cells attached to the decidual wall after removal of the embryo is an indication of the density of trophoblast cells surrounding each implantation site [17]. We found that the population of Pl-1-expressing cells was depleted in the decidual implantation sites containing Atrxnull embryos relative to those containing wild-type littermates (Figure 7B); this was also apparent at 7.5 dpc, as determined by immunohistochemical staining of paraffin sections of embryos in deciduas with an anti-Pl-1 antibody (Figure 7C). A TGC deficiency in the absence of Atrx is consistent with the observation that Atrx is highly expressed in giant cells surrounding wild-type 7.5 dpc embryos (Figure 5E).\nFigure 7 Trophectoderm Defect in Atrxnull Embryos\n(A) 8.5 dpc embryos were dissected from surrounding decidual tissue and observed in whole mount. The genotype of each (indicated above) was determined by PCR using DNA extracted from whole embryos after photography. In the left image, the wild-type female (three-somite stage, left) is surrounded by trophoblast (t) while the trophoblast component surrounding the Atrxnull males (at headfold/presomite [middle] and two-somite stages [right], respectively) is severely depleted. In the right image, the trophoblast has been dissected away from the embryonic region of the wild-type embryo, to reveal the small, abnormally shaped ectoplacental cone (epc) of the mutant littermates.\n(B) WMISH to detect expression of Pl-1 (a marker of TGCs) at the implantation sites in vacated deciduas that had contained 8.5 dpc wild-type (Atrx WT/WT) or Atrxnull (Atrx Δ18Δneo/Y) embryos. The genotype was determined by PCR using DNA extracted from whole embryos. TGCs are stained with Pl-1.\n(C) Paraffin sections of wild-type or Atrxnull 7.5 dpc embryos (dissected in their deciduas) were stained with an anti-Pl-1 antibody. The presence or absence of Atrx in each embryo was determined by staining adjacent sections with the anti-ATRX antibody (H-300) as in Figure 5 (unpublished data).\n(D) Examples of 5-d blastocyst outgrowth cultures. Extensive trophoblast outgrowing from the inner cell mass (icm) was observed in all genotypes. The Atrx genotype and sex of the blastocyst indicated were determined by PCR. To investigate whether the trophoblast defect was restricted to the production of secondary TGCs (produced by diploid precursors in the ectoplacental cone and derived originally from the polar trophectoderm overlying the inner cell mass of the blastocyst) or also affected the production of primary TGCs (resulting from differentiation of the mural trophectoderm of the blastocyst), blastocysts (3.5 dpc) from crosses between Atrx WT/flox females and GATA1-Cre homozygous transgenic males (Atrx WT/Y;GATA1-Cre +/+) were cultured in vitro for 5 d to monitor outgrowth of the primary trophoblast. After 5 d, individual blastocyst cultures were scored for the extent of primary trophoblast outgrowth, and the Atrx genotype and sex of the blastocyst were determined by PCR. Most blastocysts hatched from the zona pellucida within 24 h, and trophoblast cells spreading out from the inner cell mass could usually be detected within 48 h of culture. No difference was observed in the rate or extent of trophoblast outgrowth over 5 d of culture between Atrxnull blastocysts (Atrx null/Y, n = 6) and blastocysts bearing an Atrx WT allele (Atrx WT/WT, n = 6; Atrx WT/null, n = 6; Atrx WT/Y, n = 6) (examples shown in Figure 7D), suggesting that the defect specifically involves the secondary giant cell compartment. This is consistent with the observation that Atrxnull conceptuses implant successfully and survive to gastrulation. Taken together, these data suggest that loss of Atrx results in a defect in formation of the secondary trophoblast that is apparent from 7.5 dpc. Despite initiating normal organisation in the embryo proper, Atrxnull conceptuses exhibit a proliferative defect by 7.5 dpc and die by around 9.5 dpc, probably due to a nutritional deficit resulting from failure to develop a normal trophoblast."}

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PMC:1440874 / 26877-31668 JSON TXT