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.2013 Apr;140(8):1665-75.
doi: 10.1242/dev.087387. Epub 2013 Mar 13.

Tcf7l1 prepares epiblast cells in the gastrulating mouse embryo for lineage specification

Affiliations

Tcf7l1 prepares epiblast cells in the gastrulating mouse embryo for lineage specification

Jackson A Hoffman et al. Development.2013 Apr.

Abstract

The core gene regulatory network (GRN) in embryonic stem cells (ESCs) integrates activities of the pro-self-renewal factors Oct4 (Pou5f1), Sox2 and Nanog with that of an inhibitor of self-renewal, Tcf7l1 (Tcf3). The inhibitor function of Tcf7l1 causes dependence on extracellular Wnt/β-catenin signaling activity, making its embryonic role within the ESC GRN unclear. By analyzing intact mouse embryos, we demonstrate that the function of Tcf7l1 is necessary for specification of cell lineages to occur concomitantly with the elaboration of a three-dimensional body plan during gastrulation. In Tcf7l1(-/-) embryos, specification of mesoderm is delayed, effectively uncoupling it from the induction of the primitive streak. Tcf7l1 repressor activity is necessary for a rapid switch in the response of pluripotent cells to Wnt/β-catenin stimulation, from one of self-renewal to a mesoderm specification response. These results identify Tcf7l1 as a unique factor that is necessary in pluripotent cells to prepare them for lineage specification. We suggest that the role of Tcf7l1 in mammals is to inhibit the GRN to ensure the coordination of lineage specification with the dynamic cellular events occurring during gastrulation.

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Figures

Fig. 1.
Fig. 1.
Dynamic expression of pluripotency gene regulatory network components during gastrulation. (A-E″) Immunofluorescent staining from a representative transverse cryosection of a wild-type mouse embryo at the pre-streak (left), early-streak (middle) or mid-streak (right) stage of gastrulation. The position of sections within the intact embryo is illustrated insupplementary material Fig. S1A; posterior is at the top. The primitive streak (PS) region in the early- and mid-streak images is outlined (dashed line). Brachyury (A-A″, green), Tcf7l1 (B-B″, green), Oct4 (C-C″, cyan) and Sox2 (D-D″, yellow) are shown with co-staining for E-cadherin (magenta). The domain of Nanog immunoreactivity (E-E″, green) is marked by a solid yellow outline. (F-J) The levels of protein immunoreactivity in individual cells relative to their position in the epiblast, as described insupplementary material Fig. S1C. The intensity of immunoreactivity in individual epiblast nuclei (y-axis) is compared with distance from the PS (x-axis). The number of nuclei analyzed (n) and Pearson correlation coefficients (r) are listed for each.*P<10-5,**P<10-10.
Fig. 2.
Fig. 2.
Tcf7l1 is necessary for dynamic expression of pluripotency factors in the epiblast. (A) Quantitative RT-PCR assays measuringNanog andOct4 RNA levels relative toGapdh from individual early-streak mouse embryos. TheTcf7l1 genotype of each embryo is indicated beneath each bar;Tcf7l1-/- bars are black. (B) Whole-mountin situ hybridization analysis ofNanog mRNA expression in early-, mid- and late-streakTcf7l1+ andTcf7l1-/- embryos. Lateral views are shown; posterior is to the right. (C-E″) Immunofluorescent staining of transverse cryosections ofTcf7l1-/- embryos for Nanog (C-C″, green), Oct4 (D-D″, cyan) and Sox2 (E-E″, yellow). The position of sections within the intact embryo is illustrated insupplementary material Fig. S1A; posterior is at the top. The PS region in the early- and mid-streak images is outlined (dashed line). The solid yellow outline of Nanog expression inTcf7l1+ embryos (see Fig. 1E-E″) is overlaid onTcf7l1-/- embryos (C-C″) for comparison. (F-H) Quantitation of protein immunoreactivity in early-streak stageTcf7l1-/- embryos. (I-L) Immunofluorescent staining of transverse cryosections of early-streak stage Nanog-overexpressing embryos for Nanog (I, green), Oct4 (J, blue), Tcf7l1 (K, green) and Sox2 (L, yellow). (M-P) Quantitation of protein immunoreactivity in early-streak stage Nanog-overexpressing embryos. Protein immunoreactivity was quantitated as described in Fig. 1F-J andsupplementary material Fig. S1C.
Fig. 3.
Fig. 3.
Tcf7l1 is required for silencing of pluripotency factor expression and early neurectoderm specification. (A-I′) Whole-mountin situ hybridization forOct4 (A-C′),Nanog (D-E′),Otx2 (F,F′) andSox1 (G-I′) inTcf7l1+ (A-I) andTcf7l1-/- (A′-I′) mouse embryos of the indicates ages. Lateral views, except for frontal views in G,G′,H,H′,I′.
Fig. 4.
Fig. 4.
Coupling mesoderm specification to primitive streak formation requires Tcf7l1. (A)In situ hybridization forPrickle1 mRNA (top row, purple) and immunofluorescent detection of brachyury protein (bottom row, green) and E-cadherin (bottom row, magenta) in transverse sections of pre-, early- and mid-streak stage mouse embryos. The PS region is outlined (dashed line). (B-G) Each image shows assays from an individual early-streak stage embryo. Numbers (bottom right) indicate the position of each section relative to the most distal (#1) section pictured for each embryo. (B,C) Brachyury (cyan) and E-cadherin (magenta) protein immunoreactivity andPrickle1 mRNAin situ hybridization forTcf7l1+ (B) andTcf7l1-/- (C) embryos. (D,E)Mixl1 andPrickle1 mRNAin situ hybridization assays forTcf7l1+ (D) andTcf7l1-/- (E) embryos. (F) Brachyury (cyan) and E-cadherin (magenta) protein immunoreactivity andPrickle1 mRNAin situ hybridization for aTcf7l1ΔN/ΔN embryo. (G) Nanog (green), brachyury (cyan) and E-cadherin (magenta) protein immunoreactivity andPrickle1 mRNAin situ hybridization for a Nanog-overexpressing transgenic embryo.
Fig. 5.
Fig. 5.
Tcf7l1 is necessary for timely transition of ESCs to the EpiSC state. (A) Experimental design. ESCs were plated in ESC media for 24 hours and switched to EpiSC (FA) media at day 0. After 3 days in EpiSC conditions, samples were either cultured for 2 additional days or prepared for alkaline phosphatase (AP) staining, ESC colony formation assays and gene expression analysis. (B) Representative images of AP staining ofTcf7l1+/+ andTcf7l1-/- cells grown in EpiSC conditions for 3 days. The percentage of colonies with an EpiSC-like morphology is indicated. (C) Number of ESC colony-forming units (CFU) per 1000 cells after 3 days of culture in EpiSC conditions. (D) Quantitative RT-PCR assays measuring the levels of core pluripotency genes (Oct4, Nanog andSox2) and naïve state genes (Esrrb, Pecam1 andRex1) after 3 or 5 days in EpiSC conditions. Values are normalized to the level of expression of each gene inTcf7l1+/+ ESCs. Error bars indicate s.d. between biological replicates.
Fig. 6.
Fig. 6.
Tcf7l1 is necessary for the switch from self-renewal to mesoderm specification in response to Wnt/β-catenin signaling in pluripotent stem cells. (A) Experimental design. ESCs were plated in ESC media and switched to EpiSC (FA) media at day 1. The Gsk3 inhibitor CHIR99021 (CH) was added to media to activate Wnt/β-catenin signaling at either day 1 (CH1), day 2 (CH2) or day 3 (CH3). After 3 days in the CH1-3 regimens, samples were prepared for AP staining and assessment of colony morphology (B), ESC colony formation assays (C) and gene expression analysis (D,E). (B) AP staining of colonies after completing CH1-3 regimens. The percentage of colonies displaying an EpiSC morphology with low AP activity is shown. (C-E)Tcf7l1+/+ is depicted by white bars,Tcf7l1-/- by black bars. (C) Number of ESC CFU per 1000 cells after completing the CH1-3 regimens. (D) Quantitative RT-PCR assays measuring the levels of core pluripotency genes (Oct4, Nanog andSox2) and naïve state genes (Esrrb, Pecam1 andRex1) after completing CH1-3 regimens. Values are normalized to the level of expression of each gene inTcf7l1+/+ cells cultured in EpiSC media without CH. (E) Quantitative RT-PCR assays measuring the levels of the mesoderm genes brachyury (top) andMixl1 (bottom) in response to CH in ESCs (left), in cells undergoing transition (as diagramed in A) (middle), and in cells after 5 days of culture in EpiSC conditions (right). Values are normalized to the level ofGapdh expression in each sample. Error bars indicate s.d. between biological replicates.
Fig. 7.
Fig. 7.
Model depicting the effects of Tcf7l1 and Wnt/β-catenin on pluripotent cells in mice. As cells progress from naïve pluripotency (yellow; ESC, preimplantation epiblast) to a primed state (pink; EpiSC, postimplantation epiblast) they acquire the ability to form mesoderm in response to Wnt/β-catenin. Tcf7l1 mediates the transition from the naïve to the primed state. Wnt/β-catenin inhibits this transition.
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References

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