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Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

Galán A, Montaner D, Póo ME, Valbuena D, Ruiz V, Aguilar C, Dopazo J, Simón C - PLoS ONE (2010)

Bottom Line: The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage.Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed.Finally, new EGA features in human embryogenesis are presented.

View Article: PubMed Central - PubMed

Affiliation: Valencia Node of The National Stem Cell Bank, Centro de Investigación Príncipe Felipe, Valencia, Spain.

ABSTRACT
Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

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Gene expression analysis and blastomere fate.(A) List of the putative housekeeping genes common for blastomeres, and the ICM and TE cells from the blastocysts. Those genes with higher values and a low standard deviation (sd iqr <1) (values corresponding to quantile 25 with a sd iqr less than 1) were chosen as putative housekeeping genes (X axis). The maximum data score value in the microarray is 17.5 and 1.63 the minimum (Y axis). (B) Comparative analysis of ICM, stemness and TE gene markers in the single blastomeres from the 5-, 6- and 8- cell embryos (1) were compared to the ICM and TE differentiated cells (2). (1) No statistical difference (p value<0.05) was found for most markers except for RRAS2, FZD5 and TGFBR1 (underlined and in bold), which were up-regulated in 5-cell embryo blastomeres compared to the 6-cell (RRAS2), 8-cell (FZD5), and to both of them (TGFBR1). (2) Heat map representation of the significant differential expression (p value<0.05) between the 5- and 8- single blastomeres with ICM and TE. Gray indicates no significant differences. Red means an overexpression in the ICM or TE samples, and blue depicts an over-representation in the single blastomere samples. The color code of the expression level is indicated at the top of the figure. The complete data can be checked in Supplementary Table S2.
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pone-0013615-g002: Gene expression analysis and blastomere fate.(A) List of the putative housekeeping genes common for blastomeres, and the ICM and TE cells from the blastocysts. Those genes with higher values and a low standard deviation (sd iqr <1) (values corresponding to quantile 25 with a sd iqr less than 1) were chosen as putative housekeeping genes (X axis). The maximum data score value in the microarray is 17.5 and 1.63 the minimum (Y axis). (B) Comparative analysis of ICM, stemness and TE gene markers in the single blastomeres from the 5-, 6- and 8- cell embryos (1) were compared to the ICM and TE differentiated cells (2). (1) No statistical difference (p value<0.05) was found for most markers except for RRAS2, FZD5 and TGFBR1 (underlined and in bold), which were up-regulated in 5-cell embryo blastomeres compared to the 6-cell (RRAS2), 8-cell (FZD5), and to both of them (TGFBR1). (2) Heat map representation of the significant differential expression (p value<0.05) between the 5- and 8- single blastomeres with ICM and TE. Gray indicates no significant differences. Red means an overexpression in the ICM or TE samples, and blue depicts an over-representation in the single blastomere samples. The color code of the expression level is indicated at the top of the figure. The complete data can be checked in Supplementary Table S2.

Mentions: Housekeeping genes should be generally highly expressed and stable, meaning not variable, across samples. To search for genes accomplishing the first condition, we kept only those that had a gene expression measurement above the 3rd quartile of the intensity distribution in all arrays. To find those that, in addition were stable, we kept the genes having low standard deviation as well as low interquartile range (sd, iqr <1). (Figure 2A). Forty-six genes corresponding to the structural constituent of ribosome (RPL10L, RPLP1, RPS13, RPS24, RPL10A, RPS17, A_24_P375435, ENST00000359659), the cell surface (A_24_P551530, BCAM), ion channels (CYB561D1, HCN2, TPT1), the endopeptidase activity (TPSG1, CASP8), related to inflammatory response (LY86), autophagy (MAP1LC3A), DNA topological change (EXOC3L2) and chromatin (HIST3H3), cyclin-dependent protein kinase activity (A_32_P101195), and regulation of cell growth (OGFR), were identified (Figure 2A). The inclusion of traditional reference genes such as HPRT1, GAPDH and ACTB [37], [38], [39] was discarded. Furthermore, 13 human novel genes recently described as universal markers, including ARL18B, CTBP1, or ZNF207 [38] were not included in the selected list. Finally, one gene belonging to the group of genes related with ribosomal proteins, RPS24, was selected as the housekeeping gene for the validation assays in this study.


Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

Galán A, Montaner D, Póo ME, Valbuena D, Ruiz V, Aguilar C, Dopazo J, Simón C - PLoS ONE (2010)

Gene expression analysis and blastomere fate.(A) List of the putative housekeeping genes common for blastomeres, and the ICM and TE cells from the blastocysts. Those genes with higher values and a low standard deviation (sd iqr <1) (values corresponding to quantile 25 with a sd iqr less than 1) were chosen as putative housekeeping genes (X axis). The maximum data score value in the microarray is 17.5 and 1.63 the minimum (Y axis). (B) Comparative analysis of ICM, stemness and TE gene markers in the single blastomeres from the 5-, 6- and 8- cell embryos (1) were compared to the ICM and TE differentiated cells (2). (1) No statistical difference (p value<0.05) was found for most markers except for RRAS2, FZD5 and TGFBR1 (underlined and in bold), which were up-regulated in 5-cell embryo blastomeres compared to the 6-cell (RRAS2), 8-cell (FZD5), and to both of them (TGFBR1). (2) Heat map representation of the significant differential expression (p value<0.05) between the 5- and 8- single blastomeres with ICM and TE. Gray indicates no significant differences. Red means an overexpression in the ICM or TE samples, and blue depicts an over-representation in the single blastomere samples. The color code of the expression level is indicated at the top of the figure. The complete data can be checked in Supplementary Table S2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964308&req=5

pone-0013615-g002: Gene expression analysis and blastomere fate.(A) List of the putative housekeeping genes common for blastomeres, and the ICM and TE cells from the blastocysts. Those genes with higher values and a low standard deviation (sd iqr <1) (values corresponding to quantile 25 with a sd iqr less than 1) were chosen as putative housekeeping genes (X axis). The maximum data score value in the microarray is 17.5 and 1.63 the minimum (Y axis). (B) Comparative analysis of ICM, stemness and TE gene markers in the single blastomeres from the 5-, 6- and 8- cell embryos (1) were compared to the ICM and TE differentiated cells (2). (1) No statistical difference (p value<0.05) was found for most markers except for RRAS2, FZD5 and TGFBR1 (underlined and in bold), which were up-regulated in 5-cell embryo blastomeres compared to the 6-cell (RRAS2), 8-cell (FZD5), and to both of them (TGFBR1). (2) Heat map representation of the significant differential expression (p value<0.05) between the 5- and 8- single blastomeres with ICM and TE. Gray indicates no significant differences. Red means an overexpression in the ICM or TE samples, and blue depicts an over-representation in the single blastomere samples. The color code of the expression level is indicated at the top of the figure. The complete data can be checked in Supplementary Table S2.
Mentions: Housekeeping genes should be generally highly expressed and stable, meaning not variable, across samples. To search for genes accomplishing the first condition, we kept only those that had a gene expression measurement above the 3rd quartile of the intensity distribution in all arrays. To find those that, in addition were stable, we kept the genes having low standard deviation as well as low interquartile range (sd, iqr <1). (Figure 2A). Forty-six genes corresponding to the structural constituent of ribosome (RPL10L, RPLP1, RPS13, RPS24, RPL10A, RPS17, A_24_P375435, ENST00000359659), the cell surface (A_24_P551530, BCAM), ion channels (CYB561D1, HCN2, TPT1), the endopeptidase activity (TPSG1, CASP8), related to inflammatory response (LY86), autophagy (MAP1LC3A), DNA topological change (EXOC3L2) and chromatin (HIST3H3), cyclin-dependent protein kinase activity (A_32_P101195), and regulation of cell growth (OGFR), were identified (Figure 2A). The inclusion of traditional reference genes such as HPRT1, GAPDH and ACTB [37], [38], [39] was discarded. Furthermore, 13 human novel genes recently described as universal markers, including ARL18B, CTBP1, or ZNF207 [38] were not included in the selected list. Finally, one gene belonging to the group of genes related with ribosomal proteins, RPS24, was selected as the housekeeping gene for the validation assays in this study.

Bottom Line: The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage.Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed.Finally, new EGA features in human embryogenesis are presented.

View Article: PubMed Central - PubMed

Affiliation: Valencia Node of The National Stem Cell Bank, Centro de Investigación Príncipe Felipe, Valencia, Spain.

ABSTRACT
Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

Show MeSH
Related in: MedlinePlus