You are currently viewing the abstract.
View Full TextLog in to view the full text
AAAS login provides access to Science for AAAS members, and access to other journals in the Science family to users who have purchased individual subscriptions.
More options
Download and print this article for your personal scholarly, research, and educational use.
Buy a single issue of Science for just $15 USD.
Trunk formation in a dish
Building mammalian embryos from self-organizing stem cells in culture would accelerate the investigation of morphogenetic and differentiation processes that shape the body plan. Veenvliet et al. report a method for generating embryonic trunk-like structures (TLSs) with a neural tube, somites, and gut by embedding mouse embryonic stem cell aggregates in an extracellular matrix surrogate. Live imaging and comparative single-cell transcriptomics indicate that TLS formation is analogous to mouse development. TLSs therefore provide a scalable, tractable, and accessible high-throughput platform for decoding mammalian embryogenesis at a high level of resolution.
Science, this issue p. eaba4937
Structured Abstract
INTRODUCTION
Vertebrate development comprises multiple complex morphogenetic processes that shape the embryonic body plan through self-organization of pluripotent stem cells and their descendants. Because mammalian embryogenesis proceeds in utero, it is difficult to study the dynamics of these processes, including much-needed analysis at the cellular and molecular level. Various three-dimensional stem cell systems (“embryoids”) have been developed to circumvent this impediment. The most advanced models of post-implantation development achieved so far are gastruloids, mouse embryonic stem cell (mESC)–derived aggregates with organized gene expression domains but lacking proper morphogenesis.
RATIONALE
To advance the current models, we explored the usage of Matrigel, an extracellular matrix (ECM) surrogate. During embryonic development, the ECM provides essential chemical and mechanical cues. In vitro, lower percentages of Matrigel can drive complex tissue morphogenesis in organoids, which led us to use Matrigel embedding in various media conditions to achieve higher-order embryo-like architecture in mESC-derived aggregates.
RESULTS
We found that embedding of 96-hour gastruloids in 5% Matrigel is sufficient to induce the formation of highly organized “trunk-like structures” (TLSs), comprising the neural tube and bilateral somites with embryo-like polarity. This high level of self-organization was accompanied by accumulation of the matrix protein fibronectin at the Matrigel-TLS interface and the transcriptional up-regulation of fibronectin-binding integrins and other cell adhesion molecules. Chemical modulation of signaling pathways active in the developing mouse embryo [WNT and bone morphogenetic protein (BMP)] resulted in an excess of somites arranged like a “bunch of grapes.” Comparative time-resolved single-cell RNA sequencing of TLSs and embryos revealed that TLSs follow the same stepwise gene regulatory programs as the mouse embryo, comprising expression of critical developmental regulators at the right place and time. In particular, trunk precursors known as neuromesodermal progenitors displayed the highest differentiation potential and continuously contributed to neural and mesodermal tissue during TLS formation. In addition, live imaging demonstrated that the segmentation clock, required for rhythmic deposition of somites in vivo, ticks at an embryo-like pace in TLSs. Finally, a proof-of-principle experiment showed that Tbx6-knockout TLSs generate ectopic neural tubes at the expense of somite formation, mirroring the embryonic phenotype.
CONCLUSION
We showed that embedding of embryonic stem cell–derived aggregates in an ECM surrogate generates more advanced in vitro models that are formed in a process highly analogous to embryonic development. Trunk-like structures represent a powerful tool that is easily amenable to genetic, mechanical, chemical, or other modulations. As such, we expect them to facilitate in-depth analysis of molecular mechanisms and signaling networks that orchestrate embryonic development as well as studies of the ontogeny of mutant phenotypes in the culture dish. The scalable, tractable, and highly accessible nature of the TLS makes it a complementary in vitro platform for deciphering the dynamics of the molecular, cellular, and morphogenetic processes that shape the post-implantation embryo, at an unprecedented spatiotemporal resolution.
Embedding of mouse embryonic stem cell (mESC) aggregates in an extracellular matrix (ECM) enables generation of trunk-like structures (TLSs) with an in vivo–like architecture including gut, and neuromesodermal progenitor (NMP)–derived neural tube and somites. Comparative single-cell RNA sequencing revealed that TLS cell states and differentiation dynamics match those of the embryo. Chemical modulation and genetic manipulation highlight the utility of the TLS as a scalable, tractable, and accessible model for investigating mid-gestational embryogenesis. FN1, fibronectin.
Abstract
Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized “trunk-like structures” (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.
This is an article distributed under the terms of the Science Journals Default License.
