Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos.

Highlights d Stem cells generate mouse-embryo-like structures with improved potential d These structures undertake anterior visceral endoderm formation and gastrulation d Single-cell sequencing shows improved resemblance to mouse embryo

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction 1 , 2 . Recent work, however, has revealed roles for basement membrane remodelling in global tissue morphogenesis 3 – 5 . Upon implantation, the epiblast and extra-embryonic ectoderm of the mouse embryo become enveloped with a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis 6 , 7 . However, the mechanical role of the basement membrane for post-implantation embryogenesis remains unknown. Here, we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis 8 , 9 further regulates basement membrane remodelling by localizing Nodal signalling, and therefore activity of matrix metalloproteinases and basement-membrane perforations, to the posterior side of the embryo. Perforations on the posterior side are essential for primitive streak extension during gastrulation by rendering the prospective primitive streak’s basement membrane more prone to breaching. Thus spatio-temporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.

The dramatic morphogenetic remodeling of embryo architecture after implantation culminates in pro-amniotic cavity formation. Despite its key importance, how this transformation occurs remains unknown. Here, we apply high-resolution imaging of embryos developing in vivo and in vitro, spatial RNA sequencing and 3D trophoblaststem-cell models to determine the sequence and mechanisms of these remodeling events. We show that cavitation of the embryonic tissue is followed by folding of extraembryonic tissue to mediate formation of a second, extra-embryonic cavity. Concomitantly, at the boundary between embryonic and extra-embryonic tissues, a hybrid 3D rosette forms. Resolution of this rosette enables the embryonic cavity to invade the extra-embryonic tissue. Subsequently, β1-integrin signalling mediates formation of multiple extra-embryonic 3D rosettes. Podocalyxin exocytosis leads to their polarized resolution permitting extension of embryonic and extra-embryonic cavities and their fusion into a unified pro-amniotic cavity. These morphogenetic transformations of embryogenesis bring a novel mechanism for lumen expansion and fusion.

Mammalian embryogenesis is a paradigm of regulative development as mouse embryos show plasticity in the regulation of cell fate, cell number, and tissue morphogenesis. However, the mechanisms behind embryo plasticity remain largely unknown. Here, we determine how mouse embryos respond to an increase in cell numbers to regulate the timing and mechanism of embryonic morphogenesis, leading to the formation of the pro-amniotic cavity. Using embryos and embryonic stem cell aggregates of different size, we show that while proamniotic cavity formation in normal-sized embryos is achieved through basement membrane-induced polarization and exocytosis, cavity formation of increased-size embryos is delayed and achieved through apoptosis of cells that lack contact with the basement membrane. Importantly, blocking apoptosis, both genetically and pharmacologically, alters pro-amniotic cavity formation but does not affect size regulation in enlarged embryos. We conclude that the regulation of embryonic size and morphogenesis, albeit concomitant, have distinct molecular underpinnings.