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SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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Role of SOX17 in hPGCLC Specification(A) Western blot analysis of SOX17 expression of WT day 4 TNAP/NANOS3-mCherry-positive hPGCLCs (WT, TNAP+N3+), and whole SOX17 knockout day 4 embryoids. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and SOX17 KO day 4 embryoids.(C) RT-qPCR analysis of TNAP/NANOS3-mCherry FACS-sorted WT double-negative (TNAP-N3-) or -positive (TNAP+N3+) cells sorted from day 4 embryoids and whole SOX17 KO embryoids (SOX17 KO). Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(D) Immunofluorescence of day 4 embryoids derived from WT, SOX17 knockout (SOX17 KO), and from 1 to 1 mixture of WT and SOX17 KO 4i hESCs. The number of NANOS3-mCherry+ cells with or without SOX17 expression is shown. Quantification was based on seven to nine confocal images from four independent embryoids of each condition. Scale bars, 50 μm.(E and F) FACS analysis of TNAP and CD38 on day 5 embryoids derived from SOX17 knockout 4i hESCs containing SOX17 fusion construct with human glucocorticoid receptor ligand-binding domain (SOX17 KO+ SOX17 GR). Embryoids were derived in the presence (E) or absence (F) of cytokines with (Dex+) or without (Dex−) addition of dexamethasone.(G) RT-qPCR analysis of day 5 hPGCLC derived from WT and SOX17 KO (S17KO) and SOX17 KO + SOX17-GR (S17KO+S17GR) hESCs with (+) or without (−) dexamethasone (Dex) and in the presence (+) or absence (−) of cytokines. FACS-sorted NANOS3-mCherry/TNAP double-positive cells or whole embryoids (for S17KO) were used. Relative expression levels are shown with normalization to GAPDH. Error bars indicate mean ± SD from two biological replicates.(H) Model for establishment of hPGC transcription network by SOX17 and BLIMP1. SOX17 induces germ cell genes and, potentially, endoderm gene. Expression of BLIMP1, downstream of SOX17, suppresses endodermal genes, as well as mesodermal genes. As a result, the SOX17-BLIMP1 axis initiates hPGC program from competent cells upon induction by BMP signaling. The hPGC specification gene network is abrogated in the absence of SOX17 or BLIMP1.
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fig6: Role of SOX17 in hPGCLC Specification(A) Western blot analysis of SOX17 expression of WT day 4 TNAP/NANOS3-mCherry-positive hPGCLCs (WT, TNAP+N3+), and whole SOX17 knockout day 4 embryoids. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and SOX17 KO day 4 embryoids.(C) RT-qPCR analysis of TNAP/NANOS3-mCherry FACS-sorted WT double-negative (TNAP-N3-) or -positive (TNAP+N3+) cells sorted from day 4 embryoids and whole SOX17 KO embryoids (SOX17 KO). Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(D) Immunofluorescence of day 4 embryoids derived from WT, SOX17 knockout (SOX17 KO), and from 1 to 1 mixture of WT and SOX17 KO 4i hESCs. The number of NANOS3-mCherry+ cells with or without SOX17 expression is shown. Quantification was based on seven to nine confocal images from four independent embryoids of each condition. Scale bars, 50 μm.(E and F) FACS analysis of TNAP and CD38 on day 5 embryoids derived from SOX17 knockout 4i hESCs containing SOX17 fusion construct with human glucocorticoid receptor ligand-binding domain (SOX17 KO+ SOX17 GR). Embryoids were derived in the presence (E) or absence (F) of cytokines with (Dex+) or without (Dex−) addition of dexamethasone.(G) RT-qPCR analysis of day 5 hPGCLC derived from WT and SOX17 KO (S17KO) and SOX17 KO + SOX17-GR (S17KO+S17GR) hESCs with (+) or without (−) dexamethasone (Dex) and in the presence (+) or absence (−) of cytokines. FACS-sorted NANOS3-mCherry/TNAP double-positive cells or whole embryoids (for S17KO) were used. Relative expression levels are shown with normalization to GAPDH. Error bars indicate mean ± SD from two biological replicates.(H) Model for establishment of hPGC transcription network by SOX17 and BLIMP1. SOX17 induces germ cell genes and, potentially, endoderm gene. Expression of BLIMP1, downstream of SOX17, suppresses endodermal genes, as well as mesodermal genes. As a result, the SOX17-BLIMP1 axis initiates hPGC program from competent cells upon induction by BMP signaling. The hPGC specification gene network is abrogated in the absence of SOX17 or BLIMP1.

Mentions: We isolated and characterized the TNAP-positive cells by FACS and confirmed loss of BLIMP1, except for low expression of mutant transcripts (Figure 5D). These cells also showed loss of NANOS3, UTF1, and KLF4 and reduced expression of TFAP2C, DND1, OCT4, NANOG, and T (Figures 5D and S5B). In addition, they showed prominent upregulation of mesodermal/primitive streak and HOX genes, as well as endodermal genes, including GATA4, GATA6, FOXA1 HNF1β, and HNF4α (Figure 5D). By contrast, endodermal genes were not upregulated in Blimp1 mutant mouse PGCs (Kurimoto et al., 2008; Vincent et al., 2005). This suggests that BLIMP1 probably suppresses endoderm and other somatic genes, which might otherwise be induced by SOX17 and BMP signaling during hPGCLCs specification (Figure 6H). Loss of BLIMP1 and TFAP2C also caused upregulation of HOX genes in TCam-2 (Weber et al., 2010). This suggests that one of the roles of BLIMP1 is to continually suppress the somatic program during human germline development.


SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

Role of SOX17 in hPGCLC Specification(A) Western blot analysis of SOX17 expression of WT day 4 TNAP/NANOS3-mCherry-positive hPGCLCs (WT, TNAP+N3+), and whole SOX17 knockout day 4 embryoids. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and SOX17 KO day 4 embryoids.(C) RT-qPCR analysis of TNAP/NANOS3-mCherry FACS-sorted WT double-negative (TNAP-N3-) or -positive (TNAP+N3+) cells sorted from day 4 embryoids and whole SOX17 KO embryoids (SOX17 KO). Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(D) Immunofluorescence of day 4 embryoids derived from WT, SOX17 knockout (SOX17 KO), and from 1 to 1 mixture of WT and SOX17 KO 4i hESCs. The number of NANOS3-mCherry+ cells with or without SOX17 expression is shown. Quantification was based on seven to nine confocal images from four independent embryoids of each condition. Scale bars, 50 μm.(E and F) FACS analysis of TNAP and CD38 on day 5 embryoids derived from SOX17 knockout 4i hESCs containing SOX17 fusion construct with human glucocorticoid receptor ligand-binding domain (SOX17 KO+ SOX17 GR). Embryoids were derived in the presence (E) or absence (F) of cytokines with (Dex+) or without (Dex−) addition of dexamethasone.(G) RT-qPCR analysis of day 5 hPGCLC derived from WT and SOX17 KO (S17KO) and SOX17 KO + SOX17-GR (S17KO+S17GR) hESCs with (+) or without (−) dexamethasone (Dex) and in the presence (+) or absence (−) of cytokines. FACS-sorted NANOS3-mCherry/TNAP double-positive cells or whole embryoids (for S17KO) were used. Relative expression levels are shown with normalization to GAPDH. Error bars indicate mean ± SD from two biological replicates.(H) Model for establishment of hPGC transcription network by SOX17 and BLIMP1. SOX17 induces germ cell genes and, potentially, endoderm gene. Expression of BLIMP1, downstream of SOX17, suppresses endodermal genes, as well as mesodermal genes. As a result, the SOX17-BLIMP1 axis initiates hPGC program from competent cells upon induction by BMP signaling. The hPGC specification gene network is abrogated in the absence of SOX17 or BLIMP1.
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fig6: Role of SOX17 in hPGCLC Specification(A) Western blot analysis of SOX17 expression of WT day 4 TNAP/NANOS3-mCherry-positive hPGCLCs (WT, TNAP+N3+), and whole SOX17 knockout day 4 embryoids. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and SOX17 KO day 4 embryoids.(C) RT-qPCR analysis of TNAP/NANOS3-mCherry FACS-sorted WT double-negative (TNAP-N3-) or -positive (TNAP+N3+) cells sorted from day 4 embryoids and whole SOX17 KO embryoids (SOX17 KO). Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(D) Immunofluorescence of day 4 embryoids derived from WT, SOX17 knockout (SOX17 KO), and from 1 to 1 mixture of WT and SOX17 KO 4i hESCs. The number of NANOS3-mCherry+ cells with or without SOX17 expression is shown. Quantification was based on seven to nine confocal images from four independent embryoids of each condition. Scale bars, 50 μm.(E and F) FACS analysis of TNAP and CD38 on day 5 embryoids derived from SOX17 knockout 4i hESCs containing SOX17 fusion construct with human glucocorticoid receptor ligand-binding domain (SOX17 KO+ SOX17 GR). Embryoids were derived in the presence (E) or absence (F) of cytokines with (Dex+) or without (Dex−) addition of dexamethasone.(G) RT-qPCR analysis of day 5 hPGCLC derived from WT and SOX17 KO (S17KO) and SOX17 KO + SOX17-GR (S17KO+S17GR) hESCs with (+) or without (−) dexamethasone (Dex) and in the presence (+) or absence (−) of cytokines. FACS-sorted NANOS3-mCherry/TNAP double-positive cells or whole embryoids (for S17KO) were used. Relative expression levels are shown with normalization to GAPDH. Error bars indicate mean ± SD from two biological replicates.(H) Model for establishment of hPGC transcription network by SOX17 and BLIMP1. SOX17 induces germ cell genes and, potentially, endoderm gene. Expression of BLIMP1, downstream of SOX17, suppresses endodermal genes, as well as mesodermal genes. As a result, the SOX17-BLIMP1 axis initiates hPGC program from competent cells upon induction by BMP signaling. The hPGC specification gene network is abrogated in the absence of SOX17 or BLIMP1.
Mentions: We isolated and characterized the TNAP-positive cells by FACS and confirmed loss of BLIMP1, except for low expression of mutant transcripts (Figure 5D). These cells also showed loss of NANOS3, UTF1, and KLF4 and reduced expression of TFAP2C, DND1, OCT4, NANOG, and T (Figures 5D and S5B). In addition, they showed prominent upregulation of mesodermal/primitive streak and HOX genes, as well as endodermal genes, including GATA4, GATA6, FOXA1 HNF1β, and HNF4α (Figure 5D). By contrast, endodermal genes were not upregulated in Blimp1 mutant mouse PGCs (Kurimoto et al., 2008; Vincent et al., 2005). This suggests that BLIMP1 probably suppresses endoderm and other somatic genes, which might otherwise be induced by SOX17 and BMP signaling during hPGCLCs specification (Figure 6H). Loss of BLIMP1 and TFAP2C also caused upregulation of HOX genes in TCam-2 (Weber et al., 2010). This suggests that one of the roles of BLIMP1 is to continually suppress the somatic program during human germline development.

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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Related in: MedlinePlus