Highlights
- •A unified in vitro mouse embryo model derived exclusively from embryonic stem cells
- •Embryoids undergo advanced development to late headfold stages
- •Single-cell RNA sequencing shows similar transcriptional programs across lineages
Summary
Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extraembryonic lineages of the post-implantation embryo by transcription-factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation; formation of the anterior-posterior axis, brain, and a beating heart structure; and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole-embryo-like structure.
Introduction
At the time of implantation, the mouse blastocyst comprises three lineages: the epiblast (EPI), the trophectoderm (TE), and the primitive endoderm (PE) that will give rise to the embryo proper, the placenta, and the yolk sac, respectively. By using stem cells derived from these lineages, researchers have developed several in vitro modelsto recapitulate various events of post-implantation development. One approach has been to take solely mouse embryonic stem cells (ESCs) and, by applying exogenous stimuli, induce them to establish anterior-posterior polarity (
) and mimic basic body axis formation and aspects of gastrulation, somitogenesis, cardiogenesis, and neurulation (
;
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). Such so-called “gastruloids” are a powerful system and demonstrate the ability of ESCs to be directed into complex developmental programs. However, these systems fail to capture the entire complexity of signaling and morphological events along the complete body axes. This is largely because they fail to recapitulate the spatiotemporal interplay of signaling pathways between embryonic and extraembryonic tissues, which is crucial to pattern the post-implantation mouse embryo. Consequently, they do not represent complete embryonic structures and lack the overall morphological resemblance to natural post-implantation mouse embryos.
We have therefore adopted a second approach to fully model the post-implantation mouse embryo by promoting assembly of mouse ESCs with either extraembryonic trophoblast stem cells (TSCs), to direct formation of a post-implantation egg cylinder showing appropriate posterior development (
), or a mixture of TSCs and extraembryonic endoderm (XEN) stem cells to generate “ETX” embryos that develop anterior and posterior identity and gastrulation movements (
). Subsequently, by replacing XEN cells with ESCs harboring inducible Gata4 expression (iGata4 ESCs), we learned that it was possible to generate XEN cells at an earlier stage of development that could contribute to what we termed iETX embryos that were fully able to complete gastrulation movements (
). One remaining complication of the iETX embryo model was that TSCs and ESCs require different culture media, necessitating the use of undefined culture conditions and increasing the difficulty of developing embryoids in the laboratory. Therefore, we have developed an entirely ESC-based in vitro model that reconstitutes the three fundamental cell lineages of the post-implantation mouse embryo through transcription-factor-mediated reprogramming. In addition to replacing XEN cells with induced XEN cells, we now further substitute TSCs with ESCs that transiently overexpress Cdx2 upon doxycycline induction. We show that such induced TSCs could effectively replace TSCs to form embryo-like structures that we term “EiTiX-embryoids”. These EiTiX-embryoids undergo development from pre-gastrulation stages to neurulation stages, developing headfolds, brain, a beating heart structure, and extraembryonic tissues including a yolk sac and chorion. In agreement with the similar overall morphology, our single-cell, single-structure analysis reveals a robust recapitulation of cell states spanning both embryonic and extraembryonic lineages, with strikingly little variation in the overall gene expression program in these states. Yet, our approach also demonstrates that Cdx2-expressing cells can contribute to the chorion but not the ectoplacental cone (EPC) lineage in the extraembryonic ectoderm (ExE) compartment.
Results
Cdx2-induced ESCs self-assemble with Gata4-induced ESCs and ESCs into post-implantation-like mouse embryoids
Cdx2 is a key transcription factor driving TE development and its overexpression leads ESCs to transdifferentiate into TSC-like cells (
). To determine whether Cdx2-expressing ESCs could replace TSCs in generating ETiX-embryoids (formerly termed iETX-embryoids), we generated a transgenic ESC line carrying a doxycycline (Dox)-inducible Cdx2 gene. The resulting clones of iCdx2-ESCs showed a 100- to 200–fold increase in Cdx2 mRNA expression after 6 h of Dox induction (Figure S1A). From the four clones we tested, we selected the clone with the highest level of Cdx2 overexpression for subsequent experiments. This clone showed a substantial upregulation of both Cdx2 mRNA (Figure S1B) and protein, as detected by qRT-PCR and immunofluorescence, respectively, after 6 h of induction (Figure S1C). To assess the long-term effect of Cdx2 overexpression on cell fate, we compared three different types of cell aggregates: induced iCdx2 ESCs, uninduced iCdx2 ESCs, or TSCs (Figure 1A). After 3 days, we observed a significant upregulation of the TSC marker Eomes and downregulation of the ESC marker Oct4 in the aggregates of induced iCdx2 ESCs (Figures 1B, 1C, S1D, and S1E). Transcripts of the TSC markers Elf5, Eomes, and Gata3 were also upregulated in the induced iCdx2 ESC aggregates (Figures S1F–S1H). Together, these findings suggest that upon Cdx2 overexpression, iCdx2 ESCs lose their ESC identity and acquire TSC-like cell fate….
