Protocol for generating a 3D culture of epiblast stem cells.

Mouse gastrulation entails concomitant changes in cell fate, tissue shape, and embryo size. The use of a reproducible in vitro system is crucial for dissecting the mechanisms that coordinate these events. Here, we present a protocol for generating a 3D culture of epiblast stem cells (3D EpiSCs), which grow as epithelial spheroids mimicking key features of the gastrulating mouse embryonic epiblast. We describe steps for spheroid formation, growth, and passaging, followed by imaging or further downstream analyses. For complete details on the use and execution of this protocol, please refer to Sato et al.1.

Integrin signaling in pluripotent cells acts as a gatekeeper of mouse germline entry.

Primordial germ cells (PGCs) are the precursors of gametes and the sole mechanism by which animals transmit genetic information across generations. In the mouse embryo, the transcriptional and epigenetic regulation of PGC specification has been extensively characterized. However, the initial event that triggers the soma-germline segregation remains poorly understood. Here, we uncover a critical role for the basement membrane in regulating germline entry. We show that PGCs arise in a region of the mouse embryo that lacks contact with the basement membrane, and the addition of exogenous extracellular matrix (ECM) inhibits both PGC and PGC-like cell (PGCLC) specification in mouse embryos and stem cell models, respectively. Mechanistically, we demonstrate that the engagement of ß1 integrin with laminin blocks PGCLC specification by preventing the Wnt signaling-dependent down-regulation of the PGC transcriptional repressor, Otx2. In this way, the physical segregation of cells away from the basement membrane acts as a morphogenetic fate switch that controls the soma-germline bifurcation.

Non-apical mitoses contribute to cell delamination during mouse gastrulation.

During the epithelial-mesenchymal transition driving mouse embryo gastrulation, cells divide more frequently at the primitive streak, and half of those divisions happen away from the apical pole. These observations suggest that non-apical mitoses might play a role in cell delamination. We aim to uncover and challenge the molecular determinants of mitosis position in different regions of the epiblast through computational modeling and pharmacological treatments of embryos and stem cell-based epiblast spheroids. Blocking basement membrane degradation at the streak has no impact on the asymmetry in mitosis frequency and position. By contrast, disturbance of the actomyosin cytoskeleton or cell cycle dynamics elicits ectopic non-apical mitosis and shows that the streak region is characterized by local relaxation of the actomyosin cytoskeleton and less stringent regulation of cell division. These factors are essential for normal dynamics at the streak and favor cell delamination from the epiblast.

Developmental signals control chromosome segregation fidelity during pluripotency and neurogenesis by modulating replicative stress.

Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals-including WNT, BMP, and FGF-converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development.

Basal delamination during mouse gastrulation primes pluripotent cells for differentiation.

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.

Human blastoids 3.0.

Studying human embryo development is technically and ethically challenging. An improved protocol to generate human embryo-like structures (blastoids) from human pluripotent stem cells (PSCs) (Kagawa et al., 2021) offers innovative opportunities to dissect the mechanisms of human embryogenesis.

Recruitment of monocytes and mature macrophages in mouse pubic symphysis relaxation during pregnancy and postpartum recovery†.

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages in mouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy.