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OTX2 restricts entry to the mouse germline

Nature volume 562, pages 595–599 (2018)Cite this article

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Abstract

The successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast1. Induction requires BMP4 signalling to prospective PGCs2 and the intrinsic action of PGC transcription factors3,4,5,6. However, the molecular mechanisms that connect BMP4 to induction of the PGC transcription factors that are responsible for segregating PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Downregulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro markedly increases the efficiency of PGC-like cell differentiation and prolongs the period of PGC competence. In the absence of Otx2 activity, differentiation of PGC-like cells becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC transcription factor BLIMP1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that OTX2 functions repressively upstream of PGC transcription factors, acting as a roadblock to limit entry of epiblast cells to the germline to a small window in space and time, thereby ensuring correct numerical segregation of germline cells from the soma.

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Fig. 1: Otx2 expression is downregulated before expression of PGC transcription factors.
Fig. 2: Otx2−/− EpiLCs have an enhanced propensity to differentiate into PGCLCs.
Fig. 3: Otx2-null cells can access the germline independently of cytokines and BLIMP1.
Fig. 4: Otx2−/− ESCs contribute to the germline at an enhanced rate in vivo.

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Data availability

All the data sets generated or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank V. Wilson and D. O’Carroll for comments on the manuscript, V. Wilson for help with embryo staging, N. Mullin for pre-mRNA analyses, P. Moreira for help with embryo transfer, the CRM animal house staff for husbandry, F. Rossi and C. Cryer for FACS and B. Vernay for confocal assistance. This research was funded by the Medical and the Biotechnological and Biological Sciences Research Councils of the UK (I.C.), by a PRIN project from MIUR (A.S.) and by the Qilu Young Scholars Program of Shandong University (D.Y.).

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Nature thanks K. Hayashi, A. Johnson and D. Laird for their contribution to the peer review of this work.

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Author notes

  1. These authors contributed equally: Jingchao Zhang, Man Zhang

Authors and Affiliations

  1. MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh, EH16 4UU, Scotland

    Jingchao Zhang, Man Zhang, Matúš Vojtek & Ian Chambers

  2. Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, Via P. Castellino, 111, 80131, Naples, Italy

    Dario Acampora & Antonio Simeone

  3. Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, Jinan, 250012, PR China

    Detian Yuan

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Contributions

J.Z., M.Z. and I.C. conceived the project and designed experiments. A.S. and D.A. analysed Otx2-null embryos and provided reagents. D.Y. performed bioinformatics analysis. J.Z. and M.Z. performed experiments, with help from M.V. J.Z., M.Z. and I.C. analysed the data. I.C. wrote the paper with input from all authors.

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Correspondence to Man Zhang or Ian Chambers.

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Extended data figures and tables

Extended Data Fig. 1 Summary of cell lines used in this report.

Otx2lacZ/GFP and Otx2lacZ/fl ESCs have been described previously17. Summarized below are further modifications to Otx2 or Blimp1, or transgene additions in the above or wild-type backgrounds. Further schematic details illustrating the points of Cas9 modification of Otx2 or Blimp1 and genotype verification of derived cell lines are shown in Extended Data Fig. 3 and Extended Data Fig. 7, respectively.

Extended Data Fig. 2 Competence for germline entry is preceded by downregulation of OTX2 protein.

a, Representative cytospin images of OTX2, BLIMP1 and AP2γ staining using E14Tg2a aggregates after 1 day or 2 days of PGCLC differentiation; n = 2; scale bar, 100 μm. b, Whole-mount immunofluorescence of E14Tg2a aggregates after 1 (D1) or 2 days (D2) of differentiation of EpiLCs in the presence or absence of cytokines. Representative images of OTX2 and BLIMP1 are shown; n = 3; scale bar, 50 μm. c, Magnified image of the region highlighted in b; scale bar, 10 μm. d, Quantitative transcript analysis of Otx2 in E14Tg2a cultures with (n = 4) or without cytokines (n = 7) at indicated time point. Schematic illustration is shown in Fig. 1b. Expression levels are normalized to TBP; values are means ± s.d. e, Top, primers used for Otx2 pre-mRNA transcript analysis are shown relative to the primary transcript structure. Bottom, quantitative transcript analysis of Otx2 pre-mRNA at the indicated times (minutes) after changing E14Tg2a EpiLCs into PGCLC medium. Expression levels are normalized to TBP and shown relative to expression at t = 0; values are means ± s.d.; n = 3 biologically independent replicates. f, Assessing the temporal requirement of cytokine treatment for efficient PGCLC induction. Aggregates of E14Tg2a EpiLCs treated with cytokines for 1 (d0–d1), 2 (d0–d2) or 6 days (d0–d6) were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of PGCLCs differentiation; n = 3.

Extended Data Fig. 3 Independent Otx2−/− clones show enhanced PGCLC induction efficiency.

a, The gating strategies for analysing PGCLCs by flow cytometry. Cells were first gated based on the FSC (size) and SSC (complexities) scatter plot, followed by selection for singlets based on linear correlations between FSC-area and FSC-height. Live cells were then gated based on exclusion of DAPI to indicate cell membrane integrity. Live cells were then analysed for SSEA1 and CD61. Cells stained for fluorescence minus one (FMO) were used to set gates; stained and non-stained cells are also shown. b, Otx2lacZ/fl and Otx2 lacZ/- cells with the Oct4ΔPE::GFP reporter (two independent clones each) were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of PGCLC differentiation. For clone 5 and clone 1, n = 2; for clone 11 and clone 2, n = 9. c, Diagram showing the gRNA sequence (in red) and targeting strategy for generating Otx2-knockout cell lines. Red arrows represent genotyping primers used for screening clones. d, Immunoblot analysis of OTX2 protein expression in EpiLCs of E14Tg2a and three Otx2−/− clones. Experiment preformed once. e, E14Tg2a and three independent Otx2−/− clones generated by CRISPR/Cas9 were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of PGCLC differentiation. Two biologically independent experiments for clone c11, one for clone c17 and c19. f, Q-RT–PCR of epiblast markers during the time-course outlined in Fig. 1b. Expression levels are normalized to TBP; values are means ± s.d.; n = 3 biologically independent replicates.

Extended Data Fig. 4 OTX2 restricts PGC specification during the first two days of induction.

a, OTX2 immunofluorescence of Otx2lacZ/GFP::Otx2ERT2 ESCs before or after treatment with tamoxifen for 20 min; n = 2 biologically independent experiments; scale bar, 20 μm. b, Quantitative transcript analysis of T (Brachyury) during the time-course outlined in Fig. 1b in basal GK15 medium supplemented with the indicated cytokines. Expression levels are normalized to TBP; values are means ± s.d.; n = 3 biologically independent replicates. c, Quantitative transcript analysis of T (Brachyury), Hoxa1 and Hoxb1 during the time-course outlined in Fig. 1b in indicated cell lines. Expression levels are normalized to TBP; values are means from two biologically independent replicates. d, Left, scheme illustrating the strategy for induction or repression of Wnt signalling. E14Tg2a EpiLCs were aggregated in the indicated media and transcripts analysed at 0, 9 and 18 h. Right, quantitative transcript analysis of T (Brachyury), Hoxa1, Hoxb1 and Otx2 during the time-courses outlined on the left. Expression levels are normalized to TBP; values are means ± s.d.; n = 3 biologically independent replicates. e, Quantitative transcript analysis of T (Brachyury), Otx2, Blimp1 and Prdm14 during E14Tg2a differentiation in three different media conditions (GK15, without cytokines; PGCLC, GK15 with cytokines; PGCLC +XAV, GK15 with cytokines and with XAV939) at the indicated time point. Expression levels are normalized to TBP; values are means ± s.d.; n = 3 biologically independent replicates. f, Whole-mount immunofluorescence analysis of AP2γ and T (Brachyury) in E14Tg2a and Otx2lacZ/GFP day 2 (D2) PGCLC aggregates; n = 2 biological replicates; scale bar, 50 μm. g, Scheme illustrating tamoxifen administration schemes. h, Otx2lacZ/GFP::Otx2ERT2 cells were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of PGCLC differentiation following the tamoxifen treatment regime outlined (g); n = 2 biological replicates.

Extended Data Fig. 5 PGCLC differentiation of Otx2-null cells in the absence of cytokines.

a, Otx2lacZ/fl and Otx2lacZ/− cells carrying the Oct4ΔPE::GFP reporter (aggregates shown in Fig. 3a) were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of PGCLC differentiation in the absence of cytokines; n = 7. b, E14Tg2a and three independent Otx2−/− clones generated by CRISPR/Cas9 were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of PGCLC differentiation in the absence of cytokines. Two biologically independent experiments for clone c11, one for clone c17 and c19. c, Quantitative transcript analysis of mRNAs encoding PGC transcription factors during differentiation without PGCLC cytokines at indicated time point. Expression levels are normalized to TBP; values are means ± s.d.; n = 3 biologically independent replicates. d, Whole-mount immunostaining of aggregates of Otx2lacZ/GFP cells at day 2 in the absence of cytokines for OTX2, BLIMP1 and AP2γ; scale bar; 40 μm; n = 3.

Extended Data Fig. 6 Transcriptome analysis of EpiLCs and day 6 PGCLCs.

a, b, Heat map of the normalized gene expression and principal component analysis of microarray data (from three biologically independent replicates under seven different conditions) ordered by unsupervised hierarchical clustering; rows correspond to transcripts and columns to cells. Differentiations performed in the presence (+Cyt) or absence (-Cyt) of cytokines are indicated. WT, E14Tg2a; O−/−, Otx2lacZ/GFP; O−/− B−/−, Otx2lacZ/GFP; Blimp1−/−.

Extended Data Fig. 7 PGCLC induction of independent Blimp1-null cell lines.

a, Scheme showing the strategy used to generate Blimp1-knockout cell lines. A pair of gRNAs flanking Blimp1 exon5 were co-expressed to ensure complete deletion of Blimp1 exon5. Red arrows represent genotyping primer pairs used to screen clones. b, c, Blimp1-null clones used in Fig. 3 (b) or Extended Data Fig. 8d (c) were genotyped using primers indicated in a; n = 2 biologically independent replicates for both, all clones have been sequenced. d, Cells of the indicated genotypes (c) were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of aggregation in the presence of PGC induction cytokines; n = 2. e, f, Cells of the indicated genotypes (c) were assessed by flow cytometry for surface expression of SSEA1 and CD61 at day 6 of aggregation in the absence of PGC induction cytokines; n = 2.

Extended Data Fig. 8 Otx2−/−Blimp1−/− PGCLCs activate PGC markers.

a, Quantitative analysis of somatic transcripts at day 2 of PGCLC induction in the indicated cell lines. Expression levels are normalized to TBP; values are means ± s.d.; n = 4 biological replicates, each dot represents the value from one experiment. b, Quantitative analysis of PGC transcription factor transcripts at day 2 of PGCLC induction in the indicated cell lines. Expression levels are normalized to TBP; values are means ± s.d.; n = 6 biological replicates for E14Tg2A and Otx2lazZ/GFP, and 4 for Blimp1-knockout cell lines each dot represents the value from one experiment. c, Immunofluorescence staining for OCT4 and DAZL of cryo-sections of Otx2lacZ/GFP and Otx2lacZ/GFPBlimp1−/− aggregates at day 6 of PGCLC induction; scale bar, 50 μm and 20 μm; n = 2 biologically independent replicates. d, OCT4, H3K27me3 and H3K9me2 immunofluorescence analysis of cryo-sections of E14Tg2a, Otx2lacZ/GFP and Otx2lacZ/GFPBlimp1−/− aggregates at day 6 of PGCLC induction; scale bar, 50 μm; n = 2 biologically independent replicates.

Extended Data Fig. 9 OTX2 safeguards somatic lineages.

a. Representative morphologies and Oct4ΔPE::GFP expression of EpiSCs after one passage from EpiLCs (n = 3 for 1 clone of each genotype); scale bar; 200 μm. b, c, Flow cytometry analysis for surface expression of SSEA1 and CD61 at day 6 of PGCLC differentiation, initiated from EpiSCs after one passage from EpiLCs. One experiment for c5 and c1 and 6 biologically independent replicates for C11 and C2 (b); n = 6 biologically independent replicates (c). d, Quantitative transcript analysis of PGC transcription factors in the indicated cell lines. Expression levels are normalized to TBP; values are means ± s.d.; n = 3 biologically independent replicates, each dot represents the value from one experiment. e, Comparison of the frequency of degree of chimaerism (top) and the germ cell numbers (bottom, centre lines and error bars represents means ± s.d.) in E7.5 chimaeric embryos formed using wild-type or Otx2-null ESCs. P value (two-tailed unpaired t-test, 0.95 confidence intervals) is indicated. High, >70%; moderate, 30–70%; low, <30%. f, Bright-field and representative images of E7.5 chimaeric embryos formed by wild-type host embryos and GFP-labelled Otx2+/+ (n = 6), Otx2−/− (n = 9) or Otx2lacZ/GFP (n = 9) ESCs assessed for GFP and BLIMP1/SOX2 expression, with magnified images of the proximal posterior regions. The proportion of BLIMP1-positive cells expressing GFP in the embryos is indicated; scale bar, 100 μm (left), 20 μm. g, Summary of embryo aggregations.

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Extended Data Fig. 10 Otx2-null embryos exhibit increased number of PGCs.

a, b, Frontal-coronal (a) and sagittal (b) sections of wild-type and Otx2−/− E7.5 embryos stained with BLIMP1, AP2γ and Fragilis to detect PGCs. All sequential sections spanning the PGCLCs niche are shown; scale bar, 50 μm. The experiments were repeated in four wild-type and three Otx2-knockout embryos.

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Zhang, J., Zhang, M., Acampora, D. et al. OTX2 restricts entry to the mouse germline. Nature 562, 595–599 (2018). https://doi.org/10.1038/s41586-018-0581-5

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