Quantitative analysis of signaling responses during mouse primordial germ cell specification

Abstract

During early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a-/-), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.

Keywords: BMP; MAPK; Mouse embryo; Primordial germ cell; WNT.

Fig. 1.

Quantitative analysis PGCLC signaling responses. (A) Diagram depicting PGCLC differentiation protocol (Hayashi et al., 2011). (B) Confocal maximum intensity projection (MIP) of a Day 2 (D2) PGCLC aggregate. Scale bars: 100 μm. (C) Flow cytometry data from PGCLC differentiation. SSEA-1+ CD61+ cells represent PGCLCs. (D) Percentage of SSEA-1+ CD61+ PGCLCs over time. Each point represents an independent experiment (n=6) performed with four cell lines, represented as median and interquartile range. (E,H,K) Confocal MIPs of PGCLC aggregates at day 2, 4, and 6. Sb, 100 μm. (E) Aggregates immunostained for AP2γ (PGCLCs) and phosphorylated SMAD1/5/9 (pS1/5/9), a readout of BMP signaling response. (H) PGCLC differentiation of Spry4^H2BVenus reporter ESCs, that read out FGF/MAPK signaling activity. (K) PGCLC differentiation of TCF/Lef:H2B-GFP reporter ESCs, which read out WNT signaling activity. (F,J,M) Quantitative immunofluorescence of signaling responses in PGCLCs (AP2γ+) and non-PGCLCs (AP2γ−) in three cell aggregates/time point/cell line. Each point represents a single cell. Data shown as median and interquartile range. Student's t-test was performed on average fluorescence level per aggregate. (G) Quantitative immunofluorescence of signaling responses in PGCLCs (AP2γ+) and non-PGCLCs (AP2γ−) at early differentiation time points. Each point represents a single cell. Data shown as median and interquartile range. Student's t-test was performed on average fluorescence level per aggregate (12 h n=4, 24 h n=3). At 0 and 6 h time points, cells had not yet aggregated so statistics were performed on average fluorescence per field of view (6 h n=3, 0 h n=6). (I,L) Relative mean Spry4^H2BVenus (H) and TCF/Lef:H2B-GFP (K) fluorescence analyzed by flow cytometry. Data represented as mean and standard deviation and shown relative to mean fluorescence across all populations at day 0. n=3 experiments.

Fig. 2.

Quantitative analysis of signaling responses during PGC specification in vivo. (A) (i) Sagittal confocal optical section of an immunostained E7.25 embryo. Scale bar: 100 μm. Dashed line indicates plane of transverse section in adjacent panel. (ii) Confocal optical section of a transverse cryosection through the E7.25 allantois. Scale bar: 25 μm. Box demarcates region in higher magnification in lower panels. (B) Confocal image of a transverse section of the allantois indicating the different cell populations analyzed. Cells adjacent to PGCs (yellow) were categorized as PGC ‘Neighbors’ and non-adjacent cells within the allantois (blue) as ‘Other’ (cell populations were manually selected and pseudocolored for illustrative purposes). (C) Quantification of SOX2 levels in PGCs, Neighbors and Others within the E7.25 allantois. SOX2+ levels were used to define the PGC population. Student's t-test was performed on average fluorescence level in each embryo (n=3 embryos, number of cells indicated on graph). Each point represents a single cell. Data shown relative to average mean fluorescence in ‘Other’, non-PGCs and represented as median and interquartile range. (D,F,H) Sagittal confocal MIPs (left panels, Scale bar: 100 μm) and confocal optical sections of transverse cryosection through E7.25 and E7.75 allantois’ (Scale bar: 25 μm). Dashed line demarcates boundary between allantois and endoderm. (D) Embryos immunostained for pSMAD1/5/9. (F) Spry4^H2BVenus reporter embryos. (H) TCF/Lef:H2B-GFP reporter embryos. (E,G,I) Quantification of nuclear pSMAD1/5/9, Spry4^H2BVenus, and TCF/Lef:H2B-GFP levels in PGCs, Neighbors and Other cells within the E7.25 and E7.75 allantois’. Student's t-test was performed on average fluorescence level per embryo (n=3 embryos, number of cells indicated on graph). Each point represents a single cell. Data shown relative to average mean fluorescence in ‘Other’, non-PGCs and represented as median and interquartile range. Pr, proximal; Ds, distal; A, anterior; P, posterior; L, left; R, right; Epi, epiblast; HF; headfold; PS, primitive streak; End, endoderm.

Fig. 3.

Canonical BMP signaling is not necessary for PGCLC differentiation. (A) Confocal optical sections of wild-type (Bmpr1a^+/+) and Bmpr1a^−/− ESCs immunostained for pSMAD1/5/9 (pS1/5/9) after culture under standard conditions or after a 2-h treatment with 50 ng/ml BMP4. (B,C) Quantification of pSMAD1/5/9 levels in wild-type and Bmpr1a^−/− ESCs and epiblast-like cells (EpiLCs) from five distinct fields of view. Each point represents a single cell. Data represented as median and interquartile range. Student's t-test was performed on average fluorescence level in each field. n=2 replicates. (D) Confocal MIP of wild-type and Bmpr1a^−/− PGCLC aggregates at Day 2 (D2) of differentiation. Scale bar: 100 μm. (E) Quantification of pSMAD1/5/9 levels in wild-type and Bmpr1a^−/− PGCLC aggregates. Each point represents a single cell. Data represented as median and interquartile range. Student's t-test was performed on average fluorescence level per aggregate (n=3 aggregates). (F) Flow cytometry of wild-type and Bmpr1a^−/− aggregates at Day 2 of PGCLC differentiation. SSEA-1+ CD61+ cells represent PGCLCs. (G) Percentage of SSEA-1+ CD61+ PGCLCs during wild-type and Bmpr1a^−/− PGCLC differentiation. Each point represents an independent experiment (n=3). Data represented as median and interquartile range. (H) Left panel: confocal optical section of ESCs, cultured in serum and LIF, immunostained for the BMP pathway target, ID1 and the PGC marker AP2γ. Scale bar: 25 μm. Right panel: quantification of ID1 and AP2γ levels in individual cells. Quantification performed on images from five randomly selected regions. Linear regression and correlation coefficient analysis were performed (P<0.0001). Correlation coefficient indicated on graph. (I) Wild-type and Bmpr1a^−/− EpiLCs cells, lineage-labelled with a constitutive GFP, were mixed in equal ratios to form PGCLC aggregates. Confocal MIPs of PGCLC aggregates at day 2, 4, and 6 of differentiation. Scale bar: 100 μm.