Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo
- Hospital for Sick Children, Canada
- Karolinska Institutet, Sweden
- Ludwig Institute for Cancer Research, Karolinska Institutet, Sweden
- Karolinska Universitetssjukhuset, Sweden
- University Health Network, University of Toronto, Canada
- University of Toronto, Canada
- Slovak Academy of Sciences, Slovakia
Figures
Figure 1 with 1 supplement
Cdx2-eGFP is an early marker of the developing TE lineage, governed by Hippo signaling differences from the early 16 cell stage.
(A) Immunofluorescence staining against Cdx2 and eGFP in Cdx2-eGFP heterozygous embryos at different stages. Representative images of 10 8 cell, 39 16 cell, 35 32 cell and 11 64 cell embryos stained …
Figure 1—figure supplement 1
Developmental staging of Cdx2-eGFP embryos.
Cdx2-eGFP embryos were always staged based on cell number, which we determined in live embryos based on the number of Cdx2-eGFP positive cells present. An additional layer of ‘early’ and ‘late’ …
Figure 2
Single–cell RNA sequencing reveals gradual emergence of ICM and TE lineages.
(A) Experimental outline for harvesting single cells for RNA sequencing. (B–D) Principal component analysis using top 100 variable genes across all cells, where each cell is annotated for (B) …
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Figure 2—source data 1
Excel file of differentially expressed genes from SCDE analysis between ‘Cdx2-low’ and ‘Cdx2-high’ cell populations (based on PCA groupings) at the early 32 cell stage.
Top 50 ‘Cdx2-low’ genes and top 50 ‘Cdx2-high’ genes used as ICM- and TE-specific gene signature, respectively.
- https://doi.org/10.7554/eLife.22906.006
Figure 3 with 2 supplements
Different gene expression dynamics during development of ICM and TE lineages.
(A–B) Summary of the number of genes differentially expressed from SCDE analysis (A) between lineages within each developmental time point and (B) between developmental time points within each …
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Figure 3—source data 1
Excel file of differentially expressed genes from SCDE analysis between lineages within each developmental time point.
- https://doi.org/10.7554/eLife.22906.008
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Figure 3—source data 2
Excel file of differentially expressed genes from SCDE analysis between developmental time points within each lineage.
- https://doi.org/10.7554/eLife.22906.009
Figure 3—figure supplement 1
Examples of lineage and stage specific gene expression patterns identified by single-cell RNA sequencing.
Principal component analysis using top 100 variable genes across all cells, annotated for the expression level (log10 RPKM) of (A) TE-associated genes Dppa1, Ptges, Id2 and Anxa6 and (B) …
Figure 3—figure supplement 2
ICM commitment coincides with initiation of epiblast and primitive endoderm segregation.
Heatmap showing the expression level (log10 RPKM) of epiblast (EPI) and primitive endoderm (PE) markers for 64 cell ICM (33 cells) and late 32 cell ICM (18 cells). A panel of known EPI and PE …
Figure 4 with 1 supplement
Individual cells with different Cdx2-eGFP levels aggregated to host morulae show gradual and differential loss of developmental potential over time.
(A) Experimental outline of morula aggregation assay. (B) Examples of chimeras with TE, ICM and TE and ICM contributions, analyzed at E4.5 by immunofluorescence staining. (C) Plot showing all …
Figure 4—figure supplement 1
Live imaging chimera formation following single early 32 cell stage donor cell aggregation to host morula.
(A–B) Single plane snapshots from live imaging movies of chimera formation with (A) Cdx2-eGFP low and (B) Cdx2-eGFP high donor cells. To aid scoring of inside and outside positions in chimeras, …
Figure 5 with 1 supplement
Embryos reconstructed entirely from Cdx2-eGFP low or high cells loose their potential to recapitulate ICM and TE lineages at different times.
(A) Experimental outline to reconstruct embryos entirely of Cdx2-eGFP low, high or random cells. (B–E) Plots showing embryo reconstructions from single cells isolated from (B) late 16 cell, (C) …
Figure 5—figure supplement 1
Representative images of embryo reconstructions from single cells at different stages.
Single cells used to reconstruct embryos (left panel) are either Cdx2-eGFP low, high or randomly mixed. Reconstructed embryos live at embryonic day 4.5 (E4.5) (middle panel). Immunofluorescence …
Figure 6 with 2 supplements
ICM and TE progenitors show loss of responsiveness to Hippo signaling manipulation at the same time as they loose responsiveness to positional changes.
(A) Overview of Hippo signaling activation time course. Each bar represents 24 hr of 50 µM ROCKi treatment. (B) Percent of Cdx2 positive cells per embryo cultured for 24 hr in control or ROCKi …
Figure 6—figure supplement 1
Effect of ROCKi treatment on cell number and Hippo signaling.
(A) Total cell numbers in control and 50 µM ROCKi treated embryos at different stages. n indicates number of embryos analyzed. Statistical significance was calculated using t-test and significant p-v…
Figure 6—figure supplement 2
Expression of mCherry only does not influence cell fate in the embryo.
2 cell stage embryos were injected with H2O (wild-type control) or a cocktail of PB-TAC-mCherry-IRES-mCherry, PB-CAG-rtTA and PBase mRNA (mCherry control). Embryos were treated with Dox for 24 hours …
Figure 7
Graphical summary of specification and commitment of ICM and TE progenitors.
https://doi.org/10.7554/eLife.22906.022Videos
Video 1
Live imaging a single Cdx2-eGFP low donor cell from a 32 cell stage embryo aggregating with a host morula - donor cell moves in and contributes to the ICM.
Related to Figure 4.
Video 2
Live imaging a single Cdx2-eGFP high donor cell from a 32 cell stage embryo aggregating with a host morula – donor cell stays on the surface and contributes to the TE.
Related to Figure 4.
Video 3
Live imaging a single Cdx2-eGFP high donor cell from a 32 cell stage embryo aggregating with a host morula - donor cell moves in and contributes to the ICM.
Related to Figure 4.
