Plasticity of the inner cell mass in mouse blastocyst is restricted by the activity of FGF/MAPK pathway

Abstract

In order to ensure successful development, cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or inner cell mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential cell fate decisions. We revealed that TE-deprived ICMs derived from 32-cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.

Figure 1

Control 32-cell blastocysts and their ICMs. (A) E3.0 blastocyst labelled with the fluorescent microspheres (FM), (B) E3.0 ICM immediately after isolation, (C) E3.0 ICM 24 hrs after IS. Blue: Cdx2, green: Gata4, red: FM labelling outer cells, white: nuclei; right panel shows merged pictures; yellow (*) indicates blastocyst cavity, orange arrow indicates group of presumptive EPI cells. Scale: 20 μm.

Figure 2

Localisation of the progeny of inner cells derived from E3.0 blastocyst introduced into the 8-cell embryo (A–D and G) and the blastocyst (E–F and H) after in vitro culture. Blastocyst with GFP-positive inner cells localised: (A) exclusively in EPI, (B) in PE, (C) both in EPI and PE, (D) in TE, (E) in TE (Cdx2 was up-regulated according to the new position), (F) exclusively in ICM. Red: Gata4, green: GFP, blue: Cdx2, white: nuclei; right panel shows merged pictures, (G) Lineage contribution of inner cells introduced into the 8-cell embryo after 72 hrs of culture, (H) Lineage contribution of inner cells introduced into the blastocyst after 48 hrs of culture. Scale: 20 μm.

Figure 3

Development of E3.5 ICMs. (A) ICM immediately after IS, and (B) 24 hrs after IS. Blue: Oct4, green: Gata4, white: nuclei; right panel shows merged pictures, (C) Time-lapse imaging of E3.5 ICM immediately after isolation, after 12 hrs and after 24 hrs of in vitro culture (single optical sections), (D) The scheme of the ultimate PE origin: (a) Pdgfrα ^H2B-GFP-expressing cells localised on the surface of isolated ICM from the beginning of in vitro culture, (b) Pdgfrα ^H2B-GFP-expressing cells which translocated from inside to outside, (c) Pdgfrα^H2B-GFP-negative cells which up-regulated Pdgfrα ^H2B-GFP during in vitro culture and migrated outside, (d) Pdgfrα^H2B-GFP-negative cells which up-regulated Pdgfrα on the surface of ICM and maintained this position, (E) The scheme of the fate of all Pdgfrα ^H2B-GFP-expressing cells: (a’) cells which were localised on the surface of ICM, contributing to PE, (b’) cells which were initially placed inside and translocated outside, contributing to PE, (c’) cells localised inside, which down-regulated Pdgfrα ^H2B-GFP during culture, (d’) cells which underwent apoptosis. Scale: 20 μm.

Figure 4

Development of E4.5 ICMs. (A) ICM immediately after IS, and (B) 6 hrs after IS, (C) ICM treated with cytochalasin D (CD) 6 hrs after IS, (D–F) ICMs 24 hrs after IS: (D) surrounded by single layer of PE, (E) containing cavity, (F) surrounded by two layers of PE separated by cavity, (G) E4.5 blastocyst labelled with DiI dye, (H) E4.5 ICMs with DiI-marked PE layer immediately after IS and (I) 24 hrs after IS. Blue: Oct4 or Cdx2 or F-actin, green: Gata4, red: DiI, white: nuclei_; right panel shows merged pictures; yellow (*) indicates blastocyst cavity, (J) Time-lapse imaging of E4.5 ICM immediately after isolation, after 12 hrs and after 24 hrs of in vitro culture (single optical sections), (K) The scheme of the ultimate PE origin: (a) Pdgfrα ^H2B-GFP-expressing cells, which maintained their outside position, (b) Pdgfrα ^H2B-GFP-expressing cells, which translocated from inside to outside, (c) Pdgfrα^H2B-GFP-negative cells, which up-regulated Pdgfrα ^H2B-GFP expression during culture, (d) placed outside Pdgfrα^H2B-GFP-negative cells, which up-regulated Pdgfrα on the surface of ICM, (L) The scheme of the fate of all Pdgfrα ^H2B-GFP-expressing cells: (a’) cells which were localised on the surface of ICM, (b’) cells which changed their localisation from inside to outside, (c’) cells which down-regulated Pdgfrα ^H2B-GFP, (d’) cells which underwent apoptosis. (*) statistically significant difference compared to corresponding cell groups of E3.5 ICMs (p < 0.05). Scale: 20 μm.

Figure 5

Effect of FGF/MAPK inhibition on isolated E3.5 ICMs. (A) Control E3.5 ICM cultured 48 hrs, (B–D) ICMs derived from E3.5 blastocysts and afterwards cultured 48 hr hrs in 2inh: (B) ICM with the outer PE layer, (C) ICM with the outer TE layer, and (D) ICM with outer heterogeneous (PE and TE) layer, (E,F) ICMs derived from E3.5 blastocysts pre-incubated in 2inh. media from the 8-cell stage: (E) ICM with the outer TE layer, (F) Blastocyst with the TE, EPI and PE, (G) The time schedule of inhibitor treatment; red and black arrows indicate the culture periods in the presence or absence of inhibitors, respectively, (H) Composition of epithelial layer in isolated ICMs cultured 48 hrs, (I,J) Polarisation of E3.5 ICMs (isolated from embryos pre-incubated in 2inh. conditions from the 8-cell stage) immediately (I) and 24 hrs after IS (J), (K,L) Localisation of pYAP in E3.5 ICMs (isolated from embryos pre-incubated in 2inh. conditions from the 8-cell stage) immediately (K) and 24 hrs after IS (L), (M,N) Localisation of nuclear YAP in E3.5 ICMs immediately (M) and 24 hrs after IS (N); yellow (*) indicates blastocyst cavity, orange arrows indicate ICM cells devoid of pYAP signal. Scale: 20 μm.