Jul 6, 2011 - tion regulation (LZTS1 and ESPL1), 4 genes regulate G1/S transition (CCND2,. CDKN2A, RASSF1, and DBC1), and 6 genes are involved in ...
Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 July – 6 July, 2011
1 Tolmacheva et al., Rus. J. Genet., 2008, vol. 44, no. 11, pp. 1266-1271. 2 Kashevarova et al., Rus. J. Genet., 2009, vol. 45, no. 6, pp. 749-755.
COMPANY SYMPOSIUM ORIGIO COMPANY SYMPOSIUM Wednesday 6 July 2011
12:00 - 13:15
INVITED SESSION SESSION 64: EPIGENETICS AND EARLY EVENTS DURING MAMMALIAN DEVELOPMENT Wednesday 6 July 2011
O-257 Formation of distinct cell types in the mouse embryo M. Zernicka-Goetz1 University of Cambridge, Wellcome - CRC Institute, Cambridge, United Kingdom
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12:00 - 13:00
A crucial question in mammalian development is how cells of the early embryo differentiate into distinct cell types. These cell fate decisions are taken when cells undertake waves of asymmetric divisions, which generate one daughter cell on the inside and one on the outside of the embryo. Inside cells, so called inner cell mass (ICM), provide stem cell population for all cells of the future body, while outside cells differentiate into the trophectoderm, the extra-embryonic tissue with signalling properties. But how the fate of these inside and outside cells is decided and what decides whether a cell divides symmetrically or asymmetrically have remained unknown. To address both of these questions we combined live-cell lineage studies with gain and loss-of function approaches to assess the function of key cell fate-determining genes in this process. Our studies indicate that both cell polarisation and cell position regulate the expression of cell fate-determining genes. Moreover, we found that the natural heterogeneity among early embryo cells biases their fate. Such bias would maximise chances of developmental success while retaining developmental plasticity. O-258 X chromosome inactivation a key event during early development C. Patrat1, I. Okamoto1, D. Thépot3, N. Peynot3, P. Fauque4, N. Daniel3, P. Diabangouaya1, J. Renard3, V. Duranthon3, E. Heard1 1 Mammalian Developmental Epigenetics Group, CNRS UMR 3215/INSERM U934, Institut Curie 2 Université Paris Diderot – Assistance Publique Hôpitaux de Paris – Service de Biologie de la Reproduction, Hôpital Bichat-Claude Bernard, Paris, France 3 INRA UMR 1198 Biologie du Développement et de la Reproduction, Jouy en Josas Cedex France, 4 Laboratoire de Biologie de la reproduction CECOS, CHU de Dijon, Université de Bourgogne Introduction: In mammals, X-chromosome dosage compensation is achieved by inactivating one of the two X chromosomes in females. The developmental regulation of X chromosome inactivation (XCI) has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. But multiple strategies for initiating XCI could exist for mammals. The aim of the study was to analyse XCI initiation in rabbit and human pre implantation embryos. Material and Methods: We examined the expression of XIST RNA, which is the key regulatory molecule underlying X inactivation, the kinetics of epigenetic marks such as histone modifications induced by XIST RNA and the silencing of X-linked genes. Single cell techniques, involving RNA Fluorescence In Situ Hybridisation (FISH) and immunofluorescence with antibodies specific for different epigenetic marks have been used to assess the activity and epigenetic status of the X chromosome during development. Results: We show that in both rabbit and human pre-implantation embryos, Xist is not subject to imprinting and X inactivation initiates much later than in mouse embryos. Furthermore, Xist is up-regulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the ICM, in contrast with the mouse where X inactivation is strictly monoallelic from the outset. In rabbits, this triggers XCI on both X chromosomes, implying that the choice of which X chromosome will finally become inactive occurs downstream of Xist up-regulation. In humans, XCI is not triggered by the blastocyst stage, despite the up-regulation of XIST. Conclusion: Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis, probably as the regulation of processes such as XCI have to display substantial plasticity to accommodate evolutionary changes.
INVITED SESSION SESSION 65: MEMBERS’ INFORMATION SESSION Wednesday 6 July 2011
12:00 - 13:00
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Material and Methods: Cytotrophoblast (CT) and extraembryonic mesoderm (EM) of 6 miscarriages (11.5 ± 2.1 weeks) with diploid-aneuploid mosaicism and RB1 epimutations were analyzed using Infinium HumanMethylation27 BeadChip (Illumina, USA) covering 27,578 CpG-sites of 14,495 genes. Four out of 6 embryos had both disomic and trisomic cell lines. Two abortions were trisomic for chromosome 16, one for chromosome 10, and the other one for chromosome 21. One abortion was disomic for Y chromosome. The 6th embryo was mosaic for monosomy 13. Four miscarriages (9.9 ± 2.7 weeks) with normal karyotypes and 2 induced abortions (8.8 ± 1.8 weeks) were used as control. DNA methylation analysis was performed according to the manufactures recommendations. Data were analyzed using GenomeStudio Methylation Module software. DiffScore > |20| was used to determine significant differences in methylation level of each CpG-site between two samples. Genes associated with biological process “cell cycle” were selected using the Gene Ontology classification (GO:0007049, cell cycle, http://www.geneontology.org). Results: Twelve of 247 cell cycle genes present on the BeadChip were differentially methylated in CT and 7 genes in EM of diploid-aneuploid miscarriages. Two genes differentially methylated in CT participate in chromosome segregation regulation (LZTS1 and ESPL1), 4 genes regulate G1/S transition (CCND2, CDKN2A, RASSF1, and DBC1), and 6 genes are involved in apoptosis (TP73, PYCARD, BCL2, BRCA2, AHR, and VHL). Differentially methylated in EM CHFR and RCC2 regulate chromosome segregation, RASSF1 – G1/S transition, and ALOX15B, TP73, VHL, MYBL2 – apoptosis. None of these genes but LZTS1 and CDKN2A was differentially methylated in diploid miscarriages. DLG7, LZTS1, CDKN2A, and DCC were differentially methylated in their CT and EM respectively. Three out of 4 diploid miscarriages had one or both differentially methylated DLG7 and LZTS1 in CT. These genes are essential for proper M-phase progression and chromosome segregation. The only gene hypermethylated in CT of the remaining embryo was CDKN2A regulating G1/S transition. DCC methylated in EM regulates apoptosis and presumably trophoblast invasion. Conclusions: Abnormal methylation of cell cycle genes was shown in miscarriages with normal karyotype and diploid-aneuploid mosaicism. However the spectrum of genes, their combinations and processes they are involved are differ between these two groups of embryos. We hypothesized that inactivation of genes essential for chromosome segregation will lead to the origin of cells with numerical chromosome aberrations but further the abnormal cells will be arrested and eliminated as in the case of diploid miscarriages. If G1/S transition and apoptosis genes are deregulated as well (like in abortions with chromosomal mosaicism) then, apparently, it will favor abnormal cells survival and chromosomal mosaicism formation. This study was supported by grants of Federal Program N P806 and P1161 and State Contract for National Educational Center N 02.740.11.0281. References
