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investigate whether the reduced size of the Atrxnull embryos was due to an increase in apoptosis, we analysed sections of paraffin-embedded 7.5 dpc embryos by TdT-mediated dUTP nick end labeling (TUNEL) assay (Figure 6A). Very few apoptotic cells were detected in wild-type 7.5 dpc embryos. In Atrxnull embryos, a slight increase in the apoptotic population was evident. However, consistent with our observation of a grossly normal apoptotic index in Atrxnull ES cells (Figure S2), the apoptotic response observed in Atrxnull embryos was also not uniform, but was restricted to a low number of scattered TUNEL-positive cells. Since this small apoptotic response is unlikely to account fully for the dramatic size deficit observed in Atrxnull embryos, a possible proliferation defect was also investigated by immunohistochemical staining of 7.5 dpc embryo sections for the mitosis marker phosphorylated (Ser10) histone H3 [13]. Relative to the very high mitotic index observed in wild-type embryos, the proportion of mitotic cells observed in Atrxnull embryos at 7.5 dpc was dramatically reduced (Figure 6B). Taken together, these results suggest that the size deficit observed in Atrxnull embryos prior to lethality largely reflects a proliferative defect, with a minor but indirect contribution from increased apoptosis. Although a growth defect was also observed in Atrxnull ES cells (Figure 3A), in contrast to the Atrxnull embryos, the mitotic index of the ES cell population (as measured with the same antibody) was not depleted (Figure S1B). These observations suggest that the mitotic defect observed in embryos is unlikely to be a direct, cell-autonomous effect of the absence of Atrx, and is more likely to be a secondary effect resulting from the failure to develop a normal trophoblast (see below)."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T3885","span":{"begin":47,"end":51},"obj":"PR_EXT:000004503"},{"id":"T3886","span":{"begin":51,"end":55},"obj":"SO_EXT:sequence_nullness"},{"id":"T3887","span":{"begin":56,"end":63},"obj":"UBERON:0000922"},{"id":"T3888","span":{"begin":90,"end":99},"obj":"GO:0006915"},{"id":"T3889","span":{"begin":151,"end":158},"obj":"UBERON:0000922"},{"id":"T3890","span":{"begin":162,"end":165},"obj":"GO_PR_EXT:DNA_nucleotidylexotransferase"},{"id":"T3891","span":{"begin":175,"end":179},"obj":"CHEBI_EXT:dUTP"},{"id":"T3892","span":{"begin":189,"end":197},"obj":"CHEBI_SO_EXT:molecular_label_or_mark_or_tag_process"},{"id":"T3893","span":{"begin":234,"end":243},"obj":"GO_PATO_EXT:apoptotic_process_or_quality"},{"id":"T3894","span":{"begin":234,"end":249},"obj":"CL:0000445"},{"id":"T3895","span":{"begin":244,"end":249},"obj":"CL_GO_EXT:cell"},{"id":"T3896","span":{"begin":267,"end":276},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T3897","span":{"begin":285,"end":292},"obj":"UBERON:0000922"},{"id":"T3898","span":{"begin":297,"end":301},"obj":"PR_EXT:000004503"},{"id":"T3899","span":{"begin":301,"end":305},"obj":"SO_EXT:sequence_nullness"},{"id":"T3900","span":{"begin":306,"end":313},"obj":"UBERON:0000922"},{"id":"T3901","span":{"begin":340,"end":349},"obj":"GO_PATO_EXT:apoptotic_process_or_quality"},{"id":"T3902","span":{"begin":435,"end":444},"obj":"GO_PATO_EXT:apoptotic_process_or_quality"},{"id":"T3903","span":{"begin":454,"end":458},"obj":"PR_EXT:000004503"},{"id":"T3904","span":{"begin":458,"end":462},"obj":"SO_EXT:sequence_nullness"},{"id":"T3905","span":{"begin":463,"end":471},"obj":"CL:0002322"},{"id":"T3906","span":{"begin":466,"end":471},"obj":"CL_GO_EXT:cell"},{"id":"T3907","span":{"begin":489,"end":498},"obj":"GO_PATO_EXT:apoptotic_process_or_quality"},{"id":"T3908","span":{"begin":499,"end":507},"obj":"GO_EXT:reaction_or_response"},{"id":"T3946","span":{"begin":1443,"end":1450},"obj":"GO:0007067"},{"id":"T3947","span":{"begin":1464,"end":1471},"obj":"CL:0002322"},{"id":"T3948","span":{"begin":1467,"end":1471},"obj":"CL_GO_EXT:cell"},{"id":"T3949","span":{"begin":1467,"end":1482},"obj":"UBERON_EXT:cell_clump_or_cluster_or_group_or_mass_or_population"},{"id":"T3950","span":{"begin":1510,"end":1518},"obj":"GO:0042571"},{"id":"T3951","span":{"begin":1587,"end":1594},"obj":"GO:0007067"},{"id":"T3952","span":{"begin":1614,"end":1621},"obj":"UBERON:0000922"},{"id":"T3953","span":{"begin":1650,"end":1654},"obj":"CL_GO_EXT:cell"},{"id":"T3954","span":{"begin":1691,"end":1695},"obj":"PR_EXT:000004503"},{"id":"T3955","span":{"begin":1788,"end":1799},"obj":"UBERON:0000088"},{"id":"T3909","span":{"begin":520,"end":524},"obj":"PR_EXT:000004503"},{"id":"T3910","span":{"begin":524,"end":528},"obj":"SO_EXT:sequence_nullness"},{"id":"T3911","span":{"begin":529,"end":536},"obj":"UBERON:0000922"},{"id":"T3912","span":{"begin":622,"end":627},"obj":"CL_GO_EXT:cell"},{"id":"T3913","span":{"begin":646,"end":655},"obj":"GO_PATO_EXT:apoptotic_process_or_quality"},{"id":"T3914","span":{"begin":656,"end":664},"obj":"GO_EXT:reaction_or_response"},{"id":"T3915","span":{"begin":736,"end":740},"obj":"PR_EXT:000004503"},{"id":"T3916","span":{"begin":740,"end":744},"obj":"SO_EXT:sequence_nullness"},{"id":"T3917","span":{"begin":745,"end":752},"obj":"UBERON:0000922"},{"id":"T3918","span":{"begin":765,"end":778},"obj":"GO:0008283"},{"id":"T3919","span":{"begin":851,"end":857},"obj":"UBERON:0000922"},{"id":"T3920","span":{"begin":875,"end":882},"obj":"GO:0007067"},{"id":"T3921","span":{"begin":883,"end":889},"obj":"CHEBI_SO_EXT:molecular_indicator_or_label_or_marker_or_tag"},{"id":"T3922","span":{"begin":890,"end":904},"obj":"GO_PATO_EXT:phosphorylation_process_or_quality"},{"id":"T3923","span":{"begin":913,"end":920},"obj":"CHEBI:15358"},{"id":"T3924","span":{"begin":913,"end":923},"obj":"PR_EXT:000027594"},{"id":"T3925","span":{"begin":956,"end":963},"obj":"GO:0007067"},{"id":"T3926","span":{"begin":982,"end":991},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T3927","span":{"begin":992,"end":999},"obj":"UBERON:0000922"},{"id":"T3928","span":{"begin":1019,"end":1026},"obj":"GO:0007067"},{"id":"T3929","span":{"begin":1027,"end":1032},"obj":"CL_GO_EXT:cell"},{"id":"T3930","span":{"begin":1049,"end":1053},"obj":"SO_EXT:sequence_nullness"},{"id":"T3931","span":{"begin":1054,"end":1061},"obj":"UBERON:0000922"},{"id":"T3932","span":{"begin":1183,"end":1187},"obj":"PR_EXT:000004503"},{"id":"T3933","span":{"begin":1187,"end":1191},"obj":"SO_EXT:sequence_nullness"},{"id":"T3934","span":{"begin":1192,"end":1199},"obj":"UBERON:0000922"},{"id":"T3935","span":{"begin":1209,"end":1218},"obj":"GO_EXT:fatality_or_lethality"},{"id":"T3936","span":{"begin":1238,"end":1251},"obj":"GO_PATO_EXT:cell_proliferation_or_proliferativity"},{"id":"T3937","span":{"begin":1314,"end":1323},"obj":"GO:0006915"},{"id":"T3938","span":{"begin":1336,"end":1342},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T3939","span":{"begin":1371,"end":1375},"obj":"PR_EXT:000004503"},{"id":"T3940","span":{"begin":1375,"end":1379},"obj":"SO_EXT:sequence_nullness"},{"id":"T3941","span":{"begin":1380,"end":1388},"obj":"CL:0002322"},{"id":"T3942","span":{"begin":1383,"end":1388},"obj":"CL_GO_EXT:cell"},{"id":"T3943","span":{"begin":1421,"end":1425},"obj":"PR_EXT:000004503"},{"id":"T3944","span":{"begin":1425,"end":1429},"obj":"SO_EXT:sequence_nullness"},{"id":"T3945","span":{"begin":1430,"end":1437},"obj":"UBERON:0000922"}],"text":"To investigate whether the reduced size of the Atrxnull embryos was due to an increase in apoptosis, we analysed sections of paraffin-embedded 7.5 dpc embryos by TdT-mediated dUTP nick end labeling (TUNEL) assay (Figure 6A). Very few apoptotic cells were detected in wild-type 7.5 dpc embryos. In Atrxnull embryos, a slight increase in the apoptotic population was evident. However, consistent with our observation of a grossly normal apoptotic index in Atrxnull ES cells (Figure S2), the apoptotic response observed in Atrxnull embryos was also not uniform, but was restricted to a low number of scattered TUNEL-positive cells. Since this small apoptotic response is unlikely to account fully for the dramatic size deficit observed in Atrxnull embryos, a possible proliferation defect was also investigated by immunohistochemical staining of 7.5 dpc embryo sections for the mitosis marker phosphorylated (Ser10) histone H3 [13]. Relative to the very high mitotic index observed in wild-type embryos, the proportion of mitotic cells observed in Atrxnull embryos at 7.5 dpc was dramatically reduced (Figure 6B). Taken together, these results suggest that the size deficit observed in Atrxnull embryos prior to lethality largely reflects a proliferative defect, with a minor but indirect contribution from increased apoptosis. Although a growth defect was also observed in Atrxnull ES cells (Figure 3A), in contrast to the Atrxnull embryos, the mitotic index of the ES cell population (as measured with the same antibody) was not depleted (Figure S1B). These observations suggest that the mitotic defect observed in embryos is unlikely to be a direct, cell-autonomous effect of the absence of Atrx, and is more likely to be a secondary effect resulting from the failure to develop a normal trophoblast (see below)."}

{"project":"2_test","denotations":[{"id":"16628246-206429-85799755","span":{"begin":925,"end":927},"obj":"206429"},{"id":"T92068","span":{"begin":925,"end":927},"obj":"206429"}],"text":"To investigate whether the reduced size of the Atrxnull embryos was due to an increase in apoptosis, we analysed sections of paraffin-embedded 7.5 dpc embryos by TdT-mediated dUTP nick end labeling (TUNEL) assay (Figure 6A). Very few apoptotic cells were detected in wild-type 7.5 dpc embryos. In Atrxnull embryos, a slight increase in the apoptotic population was evident. However, consistent with our observation of a grossly normal apoptotic index in Atrxnull ES cells (Figure S2), the apoptotic response observed in Atrxnull embryos was also not uniform, but was restricted to a low number of scattered TUNEL-positive cells. Since this small apoptotic response is unlikely to account fully for the dramatic size deficit observed in Atrxnull embryos, a possible proliferation defect was also investigated by immunohistochemical staining of 7.5 dpc embryo sections for the mitosis marker phosphorylated (Ser10) histone H3 [13]. Relative to the very high mitotic index observed in wild-type embryos, the proportion of mitotic cells observed in Atrxnull embryos at 7.5 dpc was dramatically reduced (Figure 6B). Taken together, these results suggest that the size deficit observed in Atrxnull embryos prior to lethality largely reflects a proliferative defect, with a minor but indirect contribution from increased apoptosis. Although a growth defect was also observed in Atrxnull ES cells (Figure 3A), in contrast to the Atrxnull embryos, the mitotic index of the ES cell population (as measured with the same antibody) was not depleted (Figure S1B). These observations suggest that the mitotic defect observed in embryos is unlikely to be a direct, cell-autonomous effect of the absence of Atrx, and is more likely to be a secondary effect resulting from the failure to develop a normal trophoblast (see below)."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T3636","span":{"begin":47,"end":51},"obj":"PR:000004503"},{"id":"T3637","span":{"begin":56,"end":63},"obj":"UBERON:0000922"},{"id":"T3638","span":{"begin":90,"end":99},"obj":"GO:0006915"},{"id":"T3639","span":{"begin":151,"end":158},"obj":"UBERON:0000922"},{"id":"T3640","span":{"begin":234,"end":249},"obj":"CL:0000445"},{"id":"T3641","span":{"begin":285,"end":292},"obj":"UBERON:0000922"},{"id":"T3642","span":{"begin":297,"end":301},"obj":"PR:000004503"},{"id":"T3643","span":{"begin":306,"end":313},"obj":"UBERON:0000922"},{"id":"T3644","span":{"begin":454,"end":458},"obj":"PR:000004503"},{"id":"T3645","span":{"begin":463,"end":471},"obj":"CL:0002322"},{"id":"T3646","span":{"begin":520,"end":524},"obj":"PR:000004503"},{"id":"T3647","span":{"begin":529,"end":536},"obj":"UBERON:0000922"},{"id":"T3648","span":{"begin":736,"end":740},"obj":"PR:000004503"},{"id":"T3649","span":{"begin":745,"end":752},"obj":"UBERON:0000922"},{"id":"T3650","span":{"begin":765,"end":778},"obj":"GO:0008283"},{"id":"T3651","span":{"begin":851,"end":857},"obj":"UBERON:0000922"},{"id":"T3652","span":{"begin":875,"end":882},"obj":"GO:0007067"},{"id":"T3653","span":{"begin":913,"end":920},"obj":"CHEBI:15358"},{"id":"T3654","span":{"begin":913,"end":923},"obj":"PR:000027594"},{"id":"T3655","span":{"begin":956,"end":963},"obj":"GO:0007067"},{"id":"T3656","span":{"begin":992,"end":999},"obj":"UBERON:0000922"},{"id":"T3657","span":{"begin":1019,"end":1026},"obj":"GO:0007067"},{"id":"T3658","span":{"begin":1054,"end":1061},"obj":"UBERON:0000922"},{"id":"T3659","span":{"begin":1183,"end":1187},"obj":"PR:000004503"},{"id":"T3660","span":{"begin":1192,"end":1199},"obj":"UBERON:0000922"},{"id":"T3661","span":{"begin":1314,"end":1323},"obj":"GO:0006915"},{"id":"T3662","span":{"begin":1371,"end":1375},"obj":"PR:000004503"},{"id":"T3663","span":{"begin":1380,"end":1388},"obj":"CL:0002322"},{"id":"T3664","span":{"begin":1421,"end":1425},"obj":"PR:000004503"},{"id":"T3665","span":{"begin":1430,"end":1437},"obj":"UBERON:0000922"},{"id":"T3666","span":{"begin":1443,"end":1450},"obj":"GO:0007067"},{"id":"T3667","span":{"begin":1464,"end":1471},"obj":"CL:0002322"},{"id":"T3668","span":{"begin":1510,"end":1518},"obj":"GO:0042571"},{"id":"T3669","span":{"begin":1587,"end":1594},"obj":"GO:0007067"},{"id":"T3670","span":{"begin":1614,"end":1621},"obj":"UBERON:0000922"},{"id":"T3671","span":{"begin":1691,"end":1695},"obj":"PR:000004503"},{"id":"T3672","span":{"begin":1788,"end":1799},"obj":"UBERON:0000088"}],"text":"To investigate whether the reduced size of the Atrxnull embryos was due to an increase in apoptosis, we analysed sections of paraffin-embedded 7.5 dpc embryos by TdT-mediated dUTP nick end labeling (TUNEL) assay (Figure 6A). Very few apoptotic cells were detected in wild-type 7.5 dpc embryos. In Atrxnull embryos, a slight increase in the apoptotic population was evident. However, consistent with our observation of a grossly normal apoptotic index in Atrxnull ES cells (Figure S2), the apoptotic response observed in Atrxnull embryos was also not uniform, but was restricted to a low number of scattered TUNEL-positive cells. Since this small apoptotic response is unlikely to account fully for the dramatic size deficit observed in Atrxnull embryos, a possible proliferation defect was also investigated by immunohistochemical staining of 7.5 dpc embryo sections for the mitosis marker phosphorylated (Ser10) histone H3 [13]. Relative to the very high mitotic index observed in wild-type embryos, the proportion of mitotic cells observed in Atrxnull embryos at 7.5 dpc was dramatically reduced (Figure 6B). Taken together, these results suggest that the size deficit observed in Atrxnull embryos prior to lethality largely reflects a proliferative defect, with a minor but indirect contribution from increased apoptosis. Although a growth defect was also observed in Atrxnull ES cells (Figure 3A), in contrast to the Atrxnull embryos, the mitotic index of the ES cell population (as measured with the same antibody) was not depleted (Figure S1B). These observations suggest that the mitotic defect observed in embryos is unlikely to be a direct, cell-autonomous effect of the absence of Atrx, and is more likely to be a secondary effect resulting from the failure to develop a normal trophoblast (see below)."}