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Dissecting the first transcriptional divergence during human embryonic development.

Bai Q, Assou S, Haouzi D, Ramirez JM, Monzo C, Becker F, Gerbal-Chaloin S, Hamamah S, De Vos J - Stem Cell Rev (2012)

Bottom Line: Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression.A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta.The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1040, Montpellier, 34000, France.

ABSTRACT
The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.

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Induction of the TE core transcriptional circuitry induction is recapitulated during in vitro differentiation of pluripotent stem cells into trophoblast cells. a Trophoblast differentiation of pluripotent stem cells was induced by adding BMP4. Morphological changes of the hESC line HD83 cultured on Matrigel in MEF-conditioned medium after 3 days in the presence of 10 ng/mL FGF2 (left panel, negative control) or 10 ng/mL BMP4 (middle panel), and after 12 days with 10 ng/mL BMP4. Scale bar is 50 μm. b Immunofluorescence analysis showing the nuclear expression of GATA3 in HD83 cells after 5 days with BMP4. Scale bar is 10 μm. c After 5 days in the presence of 10 ng/mL BMP4, the hESC line HD83 and the iPS cell line M4C2 displayed increased expression of known trophoblast markers (CGA, KRT18 and CDX2) and decreased expression of the pluripotency marker OCT4. Expression changes were calculated by normalizing the gene expression first to the expression of the housekeeping gene GAPDH and then to gene expression in FGF2-treated control cells. * p < 0.05 and dashed line indicates the 1 fold change level. d The expression of GATA2, GATA3, GCM1, TB63, TFAP2C, CEBPA, PPP1R13L and PITX2 (TE core transcriptional regulatory circuitry) was significantly up-regulated in differentiated hESC and iPS cells (5 days with BMP4). * p < 0.05 and dashed line indicates the 1-fold change level
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Fig6: Induction of the TE core transcriptional circuitry induction is recapitulated during in vitro differentiation of pluripotent stem cells into trophoblast cells. a Trophoblast differentiation of pluripotent stem cells was induced by adding BMP4. Morphological changes of the hESC line HD83 cultured on Matrigel in MEF-conditioned medium after 3 days in the presence of 10 ng/mL FGF2 (left panel, negative control) or 10 ng/mL BMP4 (middle panel), and after 12 days with 10 ng/mL BMP4. Scale bar is 50 μm. b Immunofluorescence analysis showing the nuclear expression of GATA3 in HD83 cells after 5 days with BMP4. Scale bar is 10 μm. c After 5 days in the presence of 10 ng/mL BMP4, the hESC line HD83 and the iPS cell line M4C2 displayed increased expression of known trophoblast markers (CGA, KRT18 and CDX2) and decreased expression of the pluripotency marker OCT4. Expression changes were calculated by normalizing the gene expression first to the expression of the housekeeping gene GAPDH and then to gene expression in FGF2-treated control cells. * p < 0.05 and dashed line indicates the 1 fold change level. d The expression of GATA2, GATA3, GCM1, TB63, TFAP2C, CEBPA, PPP1R13L and PITX2 (TE core transcriptional regulatory circuitry) was significantly up-regulated in differentiated hESC and iPS cells (5 days with BMP4). * p < 0.05 and dashed line indicates the 1-fold change level

Mentions: To substantiate the TE core transcriptional regulatory circuitry induction in the trophoblast lineage, we chose to challenge this network in an in vitro model of trophoblast development. Human hESCs differentiate into extra-embryonic lineages, including trophoblast cells, when cultured in the presence of BMP4 [51, 52]. Consistent with previously published results, the hESC lines H1 and HD83 and the iPS cell line M4C2 underwent morphological changes (flattened cell shape, cobblestone appearance and patches of cells expressing GATA3) from day 3 after BMP4 addition (Fig. 6a and b). In some colonies, syncytium-like structures appeared after one to two weeks (Fig. 6a). Trophoblast markers, such as CGA, CDX2 and KRT18, were induced in the BMP4-differentiating cell population as early as day 5 after BMP4 addition (Fig. 6c). In this model, we tested the expression of nine TFs of the TE core transcriptional regulatory circuitry. Indeed, we tested nine players of this network and found that eight of them were significantly induced (GATA2, GATA3, GCM1, TB63, TFAP2C, CEBPA, PPP1R13L and PITX2) (p < 0.05) during in vitro TE differentiation (Fig. 6d). Four genes of this network are known to be induced by BMP4 (Fig. 5b) and since BMP4 is essential for in vitro trophoblast differentiation of hESC [51], it could therefore play an upstream role in inducing the complete network. Collectively, these data strongly suggests that these TFs form a TE core transcriptional regulatory circuitry that plays an active role in the induction and maintenance of the gene expression program of the placental lineage. This provides a new tool to better understand the causes of early miscarriage linked to defects in the development of trophectoderm and placenta. It would be also important to investigate whether the genes of the TE core transcriptional regulatory circuitry are differentially expressed in the developing placenta in cases of spontaneous miscarriage relative to cases of therapeutic pregnancy termination.Fig. 6


Dissecting the first transcriptional divergence during human embryonic development.

Bai Q, Assou S, Haouzi D, Ramirez JM, Monzo C, Becker F, Gerbal-Chaloin S, Hamamah S, De Vos J - Stem Cell Rev (2012)

Induction of the TE core transcriptional circuitry induction is recapitulated during in vitro differentiation of pluripotent stem cells into trophoblast cells. a Trophoblast differentiation of pluripotent stem cells was induced by adding BMP4. Morphological changes of the hESC line HD83 cultured on Matrigel in MEF-conditioned medium after 3 days in the presence of 10 ng/mL FGF2 (left panel, negative control) or 10 ng/mL BMP4 (middle panel), and after 12 days with 10 ng/mL BMP4. Scale bar is 50 μm. b Immunofluorescence analysis showing the nuclear expression of GATA3 in HD83 cells after 5 days with BMP4. Scale bar is 10 μm. c After 5 days in the presence of 10 ng/mL BMP4, the hESC line HD83 and the iPS cell line M4C2 displayed increased expression of known trophoblast markers (CGA, KRT18 and CDX2) and decreased expression of the pluripotency marker OCT4. Expression changes were calculated by normalizing the gene expression first to the expression of the housekeeping gene GAPDH and then to gene expression in FGF2-treated control cells. * p < 0.05 and dashed line indicates the 1 fold change level. d The expression of GATA2, GATA3, GCM1, TB63, TFAP2C, CEBPA, PPP1R13L and PITX2 (TE core transcriptional regulatory circuitry) was significantly up-regulated in differentiated hESC and iPS cells (5 days with BMP4). * p < 0.05 and dashed line indicates the 1-fold change level
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Related In: Results  -  Collection

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Fig6: Induction of the TE core transcriptional circuitry induction is recapitulated during in vitro differentiation of pluripotent stem cells into trophoblast cells. a Trophoblast differentiation of pluripotent stem cells was induced by adding BMP4. Morphological changes of the hESC line HD83 cultured on Matrigel in MEF-conditioned medium after 3 days in the presence of 10 ng/mL FGF2 (left panel, negative control) or 10 ng/mL BMP4 (middle panel), and after 12 days with 10 ng/mL BMP4. Scale bar is 50 μm. b Immunofluorescence analysis showing the nuclear expression of GATA3 in HD83 cells after 5 days with BMP4. Scale bar is 10 μm. c After 5 days in the presence of 10 ng/mL BMP4, the hESC line HD83 and the iPS cell line M4C2 displayed increased expression of known trophoblast markers (CGA, KRT18 and CDX2) and decreased expression of the pluripotency marker OCT4. Expression changes were calculated by normalizing the gene expression first to the expression of the housekeeping gene GAPDH and then to gene expression in FGF2-treated control cells. * p < 0.05 and dashed line indicates the 1 fold change level. d The expression of GATA2, GATA3, GCM1, TB63, TFAP2C, CEBPA, PPP1R13L and PITX2 (TE core transcriptional regulatory circuitry) was significantly up-regulated in differentiated hESC and iPS cells (5 days with BMP4). * p < 0.05 and dashed line indicates the 1-fold change level
Mentions: To substantiate the TE core transcriptional regulatory circuitry induction in the trophoblast lineage, we chose to challenge this network in an in vitro model of trophoblast development. Human hESCs differentiate into extra-embryonic lineages, including trophoblast cells, when cultured in the presence of BMP4 [51, 52]. Consistent with previously published results, the hESC lines H1 and HD83 and the iPS cell line M4C2 underwent morphological changes (flattened cell shape, cobblestone appearance and patches of cells expressing GATA3) from day 3 after BMP4 addition (Fig. 6a and b). In some colonies, syncytium-like structures appeared after one to two weeks (Fig. 6a). Trophoblast markers, such as CGA, CDX2 and KRT18, were induced in the BMP4-differentiating cell population as early as day 5 after BMP4 addition (Fig. 6c). In this model, we tested the expression of nine TFs of the TE core transcriptional regulatory circuitry. Indeed, we tested nine players of this network and found that eight of them were significantly induced (GATA2, GATA3, GCM1, TB63, TFAP2C, CEBPA, PPP1R13L and PITX2) (p < 0.05) during in vitro TE differentiation (Fig. 6d). Four genes of this network are known to be induced by BMP4 (Fig. 5b) and since BMP4 is essential for in vitro trophoblast differentiation of hESC [51], it could therefore play an upstream role in inducing the complete network. Collectively, these data strongly suggests that these TFs form a TE core transcriptional regulatory circuitry that plays an active role in the induction and maintenance of the gene expression program of the placental lineage. This provides a new tool to better understand the causes of early miscarriage linked to defects in the development of trophectoderm and placenta. It would be also important to investigate whether the genes of the TE core transcriptional regulatory circuitry are differentially expressed in the developing placenta in cases of spontaneous miscarriage relative to cases of therapeutic pregnancy termination.Fig. 6

Bottom Line: Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression.A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta.The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1040, Montpellier, 34000, France.

ABSTRACT
The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.

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Related in: MedlinePlus