Live-Imaging Analysis of Epithelial Zippering During Mouse Neural Tube Closure.

Zippering is a phenomenon of tissue morphogenesis whereby fusion between opposing epithelia progresses unidirectionally over significant distances, similar to the travel of a zip fastener, to ultimately ensure closure of an opening. A comparable process can be observed during Drosophila dorsal closure and mammalian wound healing, while zippering is employed by numerous organs such as the optic fissure, palatal shelves, tracheoesophageal foregut, and presumptive genitalia to mediate tissue sealing during normal embryonic development. Particularly striking is zippering propagation during neural tube morphogenesis, where the fusion point travels extensively along the embryonic axis to ensure closure of the neural tube. Advances in time-lapse microscopy and culture conditions have opened the opportunity for successful imaging of whole-mouse embryo development over time, providing insights into the precise cellular behavior underlying zippering propagation. Studies in mouse and the ascidian Ciona have revealed the fine-tuned cell shape changes and junction remodeling which occur at the site of zippering during neural tube morphogenesis. Here, we describe a step-by-step method for imaging at single-cell resolution the process of zippering and tissue remodeling which occurs during closure of the spinal neural tube in mouse. We also provide instructions and suggestions for quantitative morphometric analysis of cell behavior during zippering progression. This procedure can be further combined with genetic mutant models (e.g., knockouts), offering the possibility of studying the dynamics of tissue fusion and zippering propagation, which underlie a wide range of open neural tube defects.

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids.

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterized endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

At the beginning of a human pregnancy, the embryo implants into the uterus lining, known as the endometrium. At this point, the endometrium transforms into a new tissue that helps the placenta to form. Problems in this transformation process are linked to pregnancy disorders, many of which can lead to implantation failure (the embryo fails to invade the endometrium altogether) or recurrent miscarriages (the embryo implants successfully, but the interface between the placenta and the endometrium subsequently breaks down). Studying the implantation of human embryos directly is difficult due to ethical and technical barriers, and animals do not perfectly mimic the human process, making it challenging to determine the causes of pregnancy disorders. However, it is likely that a form of cellular arrest called senescence, in which cells stop dividing but remain metabolically active, plays a role. Indeed, excessive senescence in the cells that make up the endometrium is associated with recurrent miscarriage, while a lack of senescence is associated with implantation failure. To study this process, Rawlings et al. developed a new laboratory model of the human endometrium by assembling two of the main cell types found in the tissue into a three-dimensional structure. When treated with hormones, these 'assembloids' successfully mimic the activity of genes in the cells of the endometrium during implantation. Rawlings et al. then exposed the assembloids to the drug dasatinib, which targets and eliminates senescent cells. This experiment showed that assembloids become very robust and static when devoid of senescent cells. Rawlings et al. then studied the interaction between embryos and assembloids using time-lapse imaging. In the absence of dasatinib treatment, cells in the assembloid migrated towards the embryo as it expanded, a process required for implantation. However, when senescent cells were eliminated using dasatinib, this movement of cells towards the embryo stopped, and the embryo failed to expand, in a situation that mimicks implantation failure. The assembloid model of the endometrium may help scientists to study endometrial defects in the lab and test potential treatments. Further work will include other endometrial cell types in the assembloids, and could help increase the reliability of the model. However, any drug treatments identified using this model will need further research into their safety and effectiveness before they can be offered to patients.

Human embryo polarization requires PLC signaling to mediate trophectoderm specification.

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre.

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.

Rethinking Human Embryo Research Policies.

It now seems technically feasible to culture human embryos beyond the "fourteen-day limit," which has the potential to increase scientific understanding of human development and perhaps improve infertility treatments. The fourteen-day limit was adopted as a compromise but subsequently has been considered an ethical line. Does it remain relevant in light of technological advances permitting embryo maturation beyond it? Should it be changed and, if so, how and why? What justifications would be necessary to expand the limit, particularly given that doing so would violate some people's moral commitments regarding human embryos? Robust stakeholder engagement preceded adoption of the fourteen-day limit and should arguably be part of efforts to reassess it. Such engagement could also consider the need for enhanced oversight of human embryo research. In the meantime, developing and implementing reliable oversight systems should help foster high-quality research and public confidence in it.

Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure.

Epithelial fusion is a key process of morphogenesis by which tissue connectivity is established between adjacent epithelial sheets. A striking and poorly understood feature of this process is "zippering," whereby a fusion point moves directionally along an organ rudiment. Here, we uncover the molecular mechanism underlying zippering during mouse spinal neural tube closure. Fusion is initiated via local activation of integrin ß1 and focal anchorage of surface ectoderm cells to a shared point of fibronectin-rich basement membrane, where the neural folds first contact each other. Surface ectoderm cells undergo proximal junction shortening, establishing a transitory semi-rosette-like structure at the zippering point that promotes juxtaposition of cells across the midline enabling fusion propagation. Tissue-specific ablation of integrin ß1 abolishes the semi-rosette formation, preventing zippering and causing spina bifida. We propose integrin-mediated anchorage as an evolutionarily conserved mechanism of general relevance for zippering closure of epithelial gaps whose disturbance can produce clinically important birth defects.

Comparative analysis of human and mouse development: From zygote to pre-gastrulation.

Development of the mammalian embryo begins with formation of the totipotent zygote during fertilization. This initial cell is able to give rise to every embryonic tissue of the developing organism as well as all extra-embryonic lineages, such as the placenta and the yolk sac, which are essential for the initial patterning and support growth of the fetus until birth. As the embryo transits from pre- to post-implantation, major structural and transcriptional changes occur within the embryonic lineage to set up the basis for the subsequent phase of gastrulation. Fine-tuned coordination of cell division, morphogenesis and differentiation is essential to ultimately promote assembly of the future fetus. Here, we review the current knowledge of mammalian development of both mouse and human focusing on morphogenetic processes leading to the onset of gastrulation, when the embryonic anterior-posterior axis becomes established and the three germ layers start to be specified.

Vangl2 disruption alters the biomechanics of late spinal neurulation leading to spina bifida in mouse embryos.

Human mutations in the planar cell polarity component VANGL2 are associated with the neural tube defect spina bifida. Homozygous Vangl2 mutation in mice prevents initiation of neural tube closure, precluding analysis of its subsequent roles in neurulation. Spinal neurulation involves rostral-to-caudal 'zippering' until completion of closure is imminent, when a caudal-to-rostral closure point, 'Closure 5', arises at the caudal-most extremity of the posterior neuropore (PNP). Here, we used Grhl3Cre to delete Vangl2 in the surface ectoderm (SE) throughout neurulation and in an increasing proportion of PNP neuroepithelial cells at late neurulation stages. This deletion impaired PNP closure after the ∼25-somite stage and resulted in caudal spina bifida in 67% of Grhl3Cre/+Vangl2Fl/Fl embryos. In the dorsal SE, Vangl2 deletion diminished rostrocaudal cell body orientation, but not directional polarisation of cell divisions. In the PNP, Vangl2 disruption diminished mediolateral polarisation of apical neuroepithelial F-actin profiles and resulted in eversion of the caudal PNP. This eversion prevented elevation of the caudal PNP neural folds, which in control embryos is associated with formation of Closure 5 around the 25-somite stage. Closure 5 formation in control embryos is associated with a reduction in mechanical stress withstood at the main zippering point, as inferred from the magnitude of neural fold separation following zippering point laser ablation. This stress accommodation did not happen in Vangl2-disrupted embryos. Thus, disruption of Vangl2-dependent planar-polarised processes in the PNP neuroepithelium and SE preclude zippering point biomechanical accommodation associated with Closure 5 formation at the completion of PNP closure.

Biomechanical coupling facilitates spinal neural tube closure in mouse embryos.

Neural tube (NT) formation in the spinal region of the mammalian embryo involves a wave of "zippering" that passes down the elongating spinal axis, uniting the neural fold tips in the dorsal midline. Failure of this closure process leads to open spina bifida, a common cause of severe neurologic disability in humans. Here, we combined a tissue-level strain-mapping workflow with laser ablation of live-imaged mouse embryos to investigate the biomechanics of mammalian spinal closure. Ablation of the zippering point at the embryonic dorsal midline causes far-reaching, rapid separation of the elevating neural folds. Strain analysis revealed tissue expansion around the zippering point after ablation, but predominant tissue constriction in the caudal and ventral neural plate zone. This zone is biomechanically coupled to the zippering point by a supracellular F-actin network, which includes an actin cable running along the neural fold tips. Pharmacologic inhibition of F-actin or laser ablation of the cable causes neural fold separation. At the most advanced somite stages, when completion of spinal closure is imminent, the cable forms a continuous ring around the neuropore, and simultaneously, a new caudal-to-rostral zippering point arises. Laser ablation of this new closure initiation point causes neural fold separation, demonstrating its biomechanical activity. Failure of spinal closure in pre-spina bifida Zic2Ku mutant embryos is associated with altered tissue biomechanics, as indicated by greater neuropore widening after ablation. Thus, this study identifies biomechanical coupling of the entire region of active spinal neurulation in the mouse embryo as a prerequisite for successful NT closure.

Regulation of cell protrusions by small GTPases during fusion of the neural folds.

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development.

Cellular basis of neuroepithelial bending during mouse spinal neural tube closure.

Bending of the neural plate at paired dorsolateral hinge points (DLHPs) is required for neural tube closure in the spinal region of the mouse embryo. As a step towards understanding the morphogenetic mechanism of DLHP development, we examined variations in neural plate cellular architecture and proliferation during closure. Neuroepithelial cells within the median hinge point (MHP) contain nuclei that are mainly basally located and undergo relatively slow proliferation, with a 7 h cell cycle length. In contrast, cells in the dorsolateral neuroepithelium, including the DLHP, exhibit nuclei distributed throughout the apico-basal axis and undergo rapid proliferation, with a 4 h cell cycle length. As the neural folds elevate, cell numbers increase to a greater extent in the dorsolateral neural plate that contacts the surface ectoderm, compared with the more ventromedial neural plate where cells contact paraxial mesoderm and notochord. This marked increase in dorsolateral cell number cannot be accounted for solely on the basis of enhanced cell proliferation in this region. We hypothesised that neuroepithelial cells may translocate in a ventral-to-dorsal direction as DLHP formation occurs, and this was confirmed by vital cell labelling in cultured embryos. The translocation of cells into the neural fold, together with its more rapid cell proliferation, leads to an increase in cell density dorsolaterally compared with the more ventromedial neural plate. These findings suggest a model in which DLHP formation may proceed through 'buckling' of the neuroepithelium at a dorso-ventral boundary marked by a change in cell-packing density.