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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 BLIMP1 in hPGCLC Specification(A) Western blot analysis of BLIMP1 and SOX17 in TNAP-positive (TNAP+) cells sorted from wild-type (WT) and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids after hPGCLC induction. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids.(C) Immunofluorscence for OCT4 and SOX17 in cryosections of WT and BLIMP1 KO day 4 and 8 embryoids. OCT4-positive cells are highlighted. Scale bar, 50 μm.(D) Expression analysis by RT-qPCR for WT TNAP/NANOS3-mCherry double-positive cells (WT; TNAP+N3+) and BLIMP1 KO TNAP single-positive cells (BLIMP1 KO; TNAP+) sorted from day 4 embryoids. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.
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fig5: Role of BLIMP1 in hPGCLC Specification(A) Western blot analysis of BLIMP1 and SOX17 in TNAP-positive (TNAP+) cells sorted from wild-type (WT) and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids after hPGCLC induction. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids.(C) Immunofluorscence for OCT4 and SOX17 in cryosections of WT and BLIMP1 KO day 4 and 8 embryoids. OCT4-positive cells are highlighted. Scale bar, 50 μm.(D) Expression analysis by RT-qPCR for WT TNAP/NANOS3-mCherry double-positive cells (WT; TNAP+N3+) and BLIMP1 KO TNAP single-positive cells (BLIMP1 KO; TNAP+) sorted from day 4 embryoids. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.

Mentions: BLIMP1 is the first and key regulator of mouse PGC, and loss of function abrogates PGC fate (Ohinata et al., 2005; Vincent et al., 2005). However, BLIMP1 expression is apparently downstream of SOX17 in hPGCLCs (Figures 4A and 4C). We examined its mechanistic role by generating BLIMP1 knockout (KO) NANOS3-mCherry hESC line (Figure S5A). These cells showed loss of BLIMP1 by western blot (Figure 5A) and immunofluorescence (Figure S5B) on day 4 of hPGCLCs induction. Notably, there was also a loss of NANOS3-mCherry-positive cells, together with a significant reduction of NANOG, OCT4, and TFAP2C expression on day 4 (Figures 5C and S5B), indicating a failure of hPGCLC specification, and all of these cells disappeared by day 8 (Figure 5C). However, we detected ∼8% of TNAP-positive cells in day 4 embryoids (Figure 5B). This observation is highly reminiscent of the effects of Blimp1 mutation on mouse PGC specification (Ohinata et al., 2005).


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 BLIMP1 in hPGCLC Specification(A) Western blot analysis of BLIMP1 and SOX17 in TNAP-positive (TNAP+) cells sorted from wild-type (WT) and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids after hPGCLC induction. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids.(C) Immunofluorscence for OCT4 and SOX17 in cryosections of WT and BLIMP1 KO day 4 and 8 embryoids. OCT4-positive cells are highlighted. Scale bar, 50 μm.(D) Expression analysis by RT-qPCR for WT TNAP/NANOS3-mCherry double-positive cells (WT; TNAP+N3+) and BLIMP1 KO TNAP single-positive cells (BLIMP1 KO; TNAP+) sorted from day 4 embryoids. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4310934&req=5

fig5: Role of BLIMP1 in hPGCLC Specification(A) Western blot analysis of BLIMP1 and SOX17 in TNAP-positive (TNAP+) cells sorted from wild-type (WT) and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids after hPGCLC induction. TUBULIN was used as loading control.(B) FACS analysis of TNAP and NANOS3-mCherry on WT and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids.(C) Immunofluorscence for OCT4 and SOX17 in cryosections of WT and BLIMP1 KO day 4 and 8 embryoids. OCT4-positive cells are highlighted. Scale bar, 50 μm.(D) Expression analysis by RT-qPCR for WT TNAP/NANOS3-mCherry double-positive cells (WT; TNAP+N3+) and BLIMP1 KO TNAP single-positive cells (BLIMP1 KO; TNAP+) sorted from day 4 embryoids. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.
Mentions: BLIMP1 is the first and key regulator of mouse PGC, and loss of function abrogates PGC fate (Ohinata et al., 2005; Vincent et al., 2005). However, BLIMP1 expression is apparently downstream of SOX17 in hPGCLCs (Figures 4A and 4C). We examined its mechanistic role by generating BLIMP1 knockout (KO) NANOS3-mCherry hESC line (Figure S5A). These cells showed loss of BLIMP1 by western blot (Figure 5A) and immunofluorescence (Figure S5B) on day 4 of hPGCLCs induction. Notably, there was also a loss of NANOS3-mCherry-positive cells, together with a significant reduction of NANOG, OCT4, and TFAP2C expression on day 4 (Figures 5C and S5B), indicating a failure of hPGCLC specification, and all of these cells disappeared by day 8 (Figure 5C). However, we detected ∼8% of TNAP-positive cells in day 4 embryoids (Figure 5B). This observation is highly reminiscent of the effects of Blimp1 mutation on mouse PGC specification (Ohinata et al., 2005).

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