Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors

Abstract

During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast.

Keywords: Epiblast stem cells; Gastrulation; Heterogeneity; Mouse; Neuromesodermal progenitors; Primitive Streak; Wnt signalling.

Fig. 1.

Undifferentiated EpiSCs contain two major subpopulations. (A) Nanog and T(Bra) immunocytochemistry in undifferentiated, wild-type EpiSCs. Graph: immunofluorescence quantitation following single cell image analysis. Numbers: percentages of cells in each quadrant. (B) dsRed2 expression in undifferentiated Tps/tb-RED EpiSCs assessed by fluorescence microscopy (left) and flow cytometry (right). (C) qPCR for indicated markers in sorted dsRed2⁺ and dsRed2^- Tps/tb-RED EpiSCs. Results are represented as log₁₀ ratio of expression versus dsRed2^- cells (left) (^*P<0.05, Student’s t-test) or dissected E8.5 PS (right). Error bars: s.e.m. (n=3). (D) Left, fluorescence analysis of dsRed2, T(Bra) and Foxa2 expression in Tps/tb-RED EpiSCs. Right, quantitation of T(Bra) and Foxa2 positivity in dsRed2⁺ and dsRed2^- populations after immunostaining of Tps/tb-RED EpiSCs and image analysis. T, T(Bra); F, Foxa2. Error bars: s.e.m. (n=3), ^*P<0.05 (chi-squared test). (E) Nes immunocytochemistry and dsRed2 fluorescence. Arrowheads: dsRed2⁺ cells. (F) Cdh2 immunocytochemistry and dsRed2 fluorescence. Asterisks, Cdh2^high cells; arrowheads, dsRed2⁺/Cdh2^low cells. (G) Top: GFP and T(Bra) immunocytochemistry in Sox1-GFP EpiSCs. Bottom left: quantification of T(Bra) and Sox1-GFP expression domains. Bottom right: fluorescence-activated cell sorting (FACS) analysis of GFP expression in self-renewing Sox1-GFP EpiSC cultures. (H) qPCR analysis of neural and early PS marker expression in sorted Sox1-GFP ⁺ and Sox1-GFP^- EpiSCs. Results are represented as log (GFP⁺/GFP^- cell) values. Error bars: s.e.m. (n=3), ^*P<0.05 (Student’s t-test). BF, Brightfield.

Fig. 2.

PS-like Tps/tb-RED EpiSCs comprise a dynamic undifferentiated and self-renewing subpopulation. (A) Oct4, Nanog and Sox2 immunocytochemistry. Right, image analysis of dsRed2 and indicated pluripotency markers (B) Cdh1 immunocytochemistry and dsRed2 fluorescence. (C) Flow cytometry-based timecourse analysis of dsRed2 expression in sorted and replated dsRed⁺ and dsRed^- populations in EpiSC conditions. (D) Numbers of sorted dsRed2⁺ and dsRed2^- cells post-sorting. (E) Oct4 immunocytochemistry and dsRed2 fluorescence at day 6 post-sorting of dsRed2^- cells. (F) Composition of colonies obtained after plating sorted dsRed2⁺ and dsRed2^- EpiSC at clonal density for 72 hours and immunohistochemistry for T(Bra) expression. Each bar in the x-axis represents a single clone. y-axis: number of T(Bra)⁺ (red) or T(Bra)^- (grey) cells/clone. Number of clones: N_dsRed+clones=127, N_dsRed-clones=136. Pie charts: overall percentages of cells of either phenotype (number of cells: N_dsRed+cells=534, N_dsRed-cells=584).

Fig. 3.

Wnt signalling confers PS characteristics upon EpiSCs. (A) FACS analysis (upper row) and dsRed2 fluorescence (lower row) of dsRed2-expressing cells in untreated Tps/tb-RED EpiSC cultures and after treatment with Wif1 (120 hours) or CHIRON99021 (CHI) (48 hours) in Activin/Fgf2. (B) Immunocytochemistry of T(Bra) expression in control untreated, Wif1- and CHI-treated Tps/tb-RED EpiSCs. (C,D) T(Bra), Sox2 (C) and Oct4 (D) immunocytochemistry in fl/fl- and Δ/Δ-β-cat EpiSCs. Graphs: image analysis of T(Bra) and Sox2 immunofluorescence. (E) qPCR analysis of indicated markers in untreated, Wif1- (left) and CHI-treated (right) Tps/tb-RED EpiSCs. Expression is represented as log₁₀ ratio versus untreated control. Error bars: s.d. (n=2) for +Wif1 and s.e.m. (n=3) for +CHI (P<0.05 for all genes shown relative to untreated EpiSCs, Student’s t-test). (F) qPCR analysis of indicated markers in fl/fl- and Δ/Δ-β-cat EpiSCs in self-renewing conditions and after 48 hours of CHI treatment. Expression is depicted as log₁₀ ratio versus fl/fl EpiSCs (-CHI). Error bars: s.e.m. (n=3). N/D, not determined. ^*P<0.05, compared with fl/fl- β-cat EpiSCs (-CHI). (G) Percentage of dsRed2-positive cells determined by flow cytometry after 48-hour inhibitor/cytokine treatment of Tps/tb-RED EpiSC. Error bars represent s.d. (n=2). (H) Immunocytochemistry of T(Bra), Sox2 and Foxa2 in CHI-treated EpiSC cultures. (I) Heatmap representing all differentially expressed genes (≥1.5-fold change) between sorted dsRed2 positive and negative fractions and CHI-treated EpiSCs. SB, SB431542; PD, PD0325901; Wnt, Wnt3a.

Fig. 4.

Tps/tb promoter activity in EpiSCs correlates with distinct differentiation choices in vitro. (A) Cardiomyocyte differentiation expressed as percentage of beating foci/total plated EBs at different time points after sorting dsRed2⁺ and dsRed2^- EpiSCs. Unsorted EBs are included as a control. Error bars represent s.e.m. (n=3). ^*P<0.05 (Student’s t-test). (B) Immunocytochemistry of Nkx2-5 expression in plated EBs derived from sorted dsRed2⁺ and dsRed2^- EpiSC fractions. (C) qPCR analysis of paraxial mesoderm and cardiac/endoderm marker expression in sorted dsRed2⁺ and dsRed2^- EpiSC-derived EBs. Values are expressed relative to undifferentiated EpiSCs. Error bars: s.e.m. (n=3). ^*P<0.05 (Student’s t-test). (D) T(Bra) and Foxa2 immunofluorescence in colonies obtained after plating sorted Tps/tb-RED EpiSCs at clonal density for 48 hours in the presence of CHI. (E) Composition of colonies obtained after plating sorted dsRed2⁺ and dsRed2^- EpiSC at clonal density for 48 hours in CHI, depicted as in Fig. 2F. Number of clones: N_dsRed+clones=126, N_dsRed-clones=130. Pie charts: overall percentages of cells of each phenotype (number of cells: N_dsRed+cells=409, N_dsRed-cells=385). (F) Proportions of T(Bra)+ cells in steady-state dsRed2⁺ and dsRed2^- EpiSC populations (data from Fig. 1D) and after sorting and plating in EpiSC (data from Fig. 2F) or CHI treatment conditions (data from Fig. 4E). C, control.

Fig. 5.

Tps/tb promoter activity in EpiSCs correlates with distinct differentiation choices in vivo. (A) Diagram showing graft sites in mid-late streak embryo (mid-posterior, MP; and anterior streak, Ant). (B) Extent of incorporation (percentage of sections containing donor cells/total sections) after grafting sorted dsRed2⁺GFP⁺, dsRed2^-GFP⁺ or unsorted GFP⁺ EpiSCs into the indicated sites. P-values were calculated using the χ² test. For Ant grafts: N_dsRed+=4, N_dsRed-=5, N_unsorted=2). For MP grafts: N_dsRed+=4, N_dsRed-=4, N_unsorted=4). ^*P<0.05; ^**P<0.01 (chi-squared test). See supplementary material Table S2 for individual embryo data. (C) Representative examples of M-LS embryo grafts (0 hours) and distribution of sorted dsRed2⁺GFP⁺ and dsRed2^-GFP⁺ EpiSCs after 24 hours embryo culture. Arrowheads, graft sites. Asterisk, clump in amniotic cavity. (D) Representative examples of donor cell incorporation (green, GFP) and differentiation (red, immunofluorescence for indicated markers). Grey, 4′,6-diamidino-2-phenylindole (DAPI) counterstain. Arrowheads, co-expressing donor cells. Note: dsRed2 expression was undetectable after 48-hour culture in the absence of doxycycline. (E) Extent of incorporation after grafting of CHI-treated GFP⁺ EpiSCs into the anterior PS of M-LS stage embryos. Note: untreated control frequencies reproduced from B (^*P<0.01; N_+CHI=4, N_unsorted=2). (F,G) Grafting of C2 EpiSCs ±CHI treatment to E8.5 embryos and culture for 24-48 hours. (F) Left, representative embryos and right, donor cell contribution. (G) Green, GFP immunofluorescence; red, indicated marker immunofluorescence in representative sections after culture; blue or grey, DAPI counterstain. Note that the images depicting NP and PXM contribution belong to the same embryo. Arrowheads, co-expressing donor cell. AL, allantois; Ant, anterior streak; AXM, axial mesoderm; DA, dorsal aorta; EN, endoderm; MP, mid posterior; NON-INT, non-integrated cell clumps; NP, neural plate; PXM/LM, paraxial/lateral mesoderm; PS, primitive streak; V/LM, ventral/lateral mesoderm.

Fig. 6.

Model of PS precursor specification in vitro. Undifferentiated EpiSC cultures contain two major mutually exclusive subpopulations: a Wnt/β-catenin-low partially committed neural-like and a PS-biased pluripotent, reversible fraction marked by slightly higher Wnt levels. Further elevation of Wnt/β-catenin signalling above this pluripotency-compatible Wnt activity threshold promotes the generation of lineage-restricted populations resembling mesendodermal (ME) and neuromesodermal (NM) precursors. Red arrows, Wnt effects.