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To implant in the uterus, the mammalian embryo first specifies two cell lineages: the pluripotent inner cell mass that forms the fetus, and the outer trophectoderm layer that forms the placenta 1 . In many organisms, asymmetrically inherited fate determinants drive lineage specification 2 , but this is not thought to be the case during early mammalian development. Here we show that intermediate filaments assembled by keratins function as asymmetrically inherited fate determinants in the mammalian embryo. Unlike F-actin or microtubules, keratins are the first major components of the cytoskeleton that display prominent cell-to-cell variability, triggered by heterogeneities in the BAF chromatin-remodelling complex. Live-embryo imaging shows that keratins become asymmetrically inherited by outer daughter cells during cell division, where they stabilize the cortex to promote apical polarization and YAP-dependent expression of CDX2, thereby specifying the first trophectoderm cells of the embryo. Together, our data reveal a mechanism by which cell-to-cell heterogeneities that appear before the segregation of the trophectoderm and the inner cell mass influence lineage fate, via differential keratin regulation, and identify an early function for intermediate filaments in development. Keratins are determinants of cell fate during mammalian embryogenesis, and are distributed asymmetrically between daughter cells during cell division.

Very little is known about the role of transcription factors DNA-binding dynamics in defining development and pluriotency. Cells in the early mouse embryo display two classes of Oct4 kinetics that define two subpopulations of cells with distinct lineage potential. Transcription factors are central to sustaining pluripotency, yet little is known about transcription factor dynamics in defining pluripotency in the early mammalian embryo. Here, we establish a fluorescence decay after photoactivation (FDAP) assay to quantitatively study the kinetic behaviour of Oct4, a key transcription factor controlling pre-implantation development in the mouse embryo. FDAP measurements reveal that each cell in a developing embryo shows one of two distinct Oct4 kinetics, before there are any morphologically distinguishable differences or outward signs of lineage patterning. The differences revealed by FDAP are due to differences in the accessibility of Oct4 to its DNA binding sites in the nucleus. Lineage tracing of the cells in the two distinct sub-populations demonstrates that the Oct4 kinetics predict lineages of the early embryo. Cells with slower Oct4 kinetics are more likely to give rise to the pluripotent cell lineage that contributes to the inner cell mass. Those with faster Oct4 kinetics contribute mostly to the extra-embryonic lineage. Our findings identify Oct4 kinetics, rather than differences in total transcription factor expression levels, as a predictive measure of developmental cell lineage patterning in the early mouse embryo.

During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity. Contractile forces are key to sorting the embryonic inner cell mass from the extraembryonic trophectoderm. Here they show that Lamin-A links changes in mechanical forces to cell fate specification, enabling Yap-Cdx2 signaling in outer, but not inner cells.

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes. Natural killer (NK) cells are assessed for various therapies, but sub-optimal cryopreservation dampens their clinical feasibility. Here the authors show that pretreating human NK cells with IL-15/IL-18 prior to cryopreservation improves NK cell post-thaw viability and functions, potentially via anti-apoptosis gene induction and granzyme B degranulation.

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of \"off-targets\", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease. Many genes working within large gene networks influence the development of heart muscle cells in humans and other animals. The activity of these genes is controlled in part by proteins called transcription factors, which bind to DNA and act as molecular switches. One transcription factor that is particularly important for the development of heart muscle cells is called NKX2-5. Mice lacking NKX2-5 have abnormal hearts and many humans who are born with congenital heart disease carry mutations in the gene that encodes this protein. Many of these mutations alter a section of the protein called the homeodomain, and therefore interfere with the ability of NKX2-5 to bind to DNA or associate with other important cardiac proteins called cofactors. However, it is not clear how such mutations alter the behaviour of NKX2-5 across all of its targets. Bouveret et al. have now used a technique called ‘DNA adenine methyltransferase identification’ to study how NKX2-5 interacts with other proteins and DNA. The experiments found that, as expected, the mutant NKX2-5 proteins were unable to associate with many of the usual gene and protein targets of normal NKX2-5. However, the mutant proteins were still able to bind to some of their usual targets, plus many other targets that the normal NKX2-5 protein was not able to bind to. A particular NKX2-5 mutant protein that the experiments analysed was missing the entire homeodomain, yet it was still able to associate with the normal NKX2-5 protein and bind to cofactors that help NKX2-5 to find its usual targets. This finding led Bouveret et al. to discover the role of a section of the NKX2-5 protein called the tyrosine-rich domain, which in the absence of the homeodomain can direct interactions of NKX2-5 with itself and its cofactors. Bouveret et al.'s findings suggest that protein cofactors of NKX2-5 help mutant NKX2-5 proteins retain some of their normal activities, but also direct the mutant proteins to abnormal gene targets, which could contribute to congenital heart disease. The next steps are to carry out experiments in animals to confirm these findings, and to understand the activities of mutant NKX2-5 and other mutant transcription factors across the whole genome. This could lead to new therapeutic approaches to treat congenital heart disease and other conditions.

The cytoskeleton — comprising actin filaments, microtubules and intermediate filaments — serves instructive roles in regulating cell function and behaviour during development. However, a key challenge in cell and developmental biology is to dissect how these different structures function and interact in vivo to build complex tissues, with the ultimate aim to understand these processes in a mammalian organism. The preimplantation mouse embryo has emerged as a primary model system for tackling this challenge. Not only does the mouse embryo share many morphological similarities with the human embryo during its initial stages of life, it also permits the combination of genetic manipulations with live-imaging approaches to study cytoskeletal dynamics directly within an intact embryonic system. These advantages have led to the discovery of novel cytoskeletal structures and mechanisms controlling lineage specification, cell–cell communication and the establishment of the first forms of tissue architecture during development. Here we highlight the diverse organization and functions of each of the three cytoskeletal filaments during the key events that shape the early mammalian embryo, and discuss how they work together to perform key developmental tasks, including cell fate specification and morphogenesis of the blastocyst. Collectively, these findings are unveiling a new picture of how cells in the early embryo dynamically remodel their cytoskeleton with unique spatial and temporal precision to drive developmental processes in the rapidly changing in vivo environment.The cytoskeleton has been extensively implicated in regulating cell function and behaviour during development. This Review analyses the functional organization of cytoskeletal components in the early mouse embryo, and discusses key roles of the cytoskeleton during early mammalian embryogenesis, including regulation of cell fate specification and morphogenesis of the blastocyst.

Unlike the mechanisms involved in the death of neuronal cell bodies, those causing the elimination of processes are not well understood owing to the lack of suitable experimental systems. As the neurotrophin receptor p75 NTR is known to restrict the growth of neuronal processes, we engineered mouse embryonic stem (ES) cells to express an Ngfr (p75 NTR ) cDNA under the control of the Mapt locus (the gene encoding tau), which begins to be active when ES cell–derived progenitors start elongating processes. This caused a progressive, synchronous degeneration of all processes, and a prospective proteomic analysis showed increased levels of the sugar-binding protein galectin-1 in the p75 NTR -engineered cells. Function-blocking galectin-1 antibodies prevented the degeneration of processes, and recombinant galectin-1 caused the processes of wild-type neurons to degenerate first, followed by the cell bodies. In vivo , the application of a glutamate receptor agonist, a maneuver known to upregulate p75 NTR , led to an increase in the amount of galectin-1 and to the degeneration of neurons and their processes in a galectin-1–dependent fashion. Section of the sciatic nerve also rapidly upregulated levels of p75 NTR and galectin-1 in terminal Schwann cells, and the elimination of nerve endings was delayed at the neuromuscular junction of mice lacking Lgals1 (the gene encoding galectin-1). These results indicate that galectin-1 actively participates in the elimination of neuronal processes after lesion, and that engineered ES cells are a useful tool for studying relevant aspects of neuronal degeneration that have been hitherto difficult to analyze.

Compaction of the preimplantation embryo is the earliest morphogenetic process essential for mammalian development, yet it remains unclear how round cells elongate to form a compacted embryo. Here, using live mouse embryo imaging, we demonstrate that cells extend long E-cadherin-dependent filopodia on to neighbouring cells, which control the cell shape changes necessary for compaction. We found that filopodia extension is tightly coordinated with cell elongation, whereas retraction occurs before cells become round again before dividing. Laser-based ablations revealed that filopodia are required to maintain elongated cell shapes. Moreover, molecular disruption of the filopodia components E-cadherin, α- and β-catenin, F-actin and myosin-X prevents cells from elongating and compacting the embryo. Finally, we show that early filopodia formation triggered by overexpressing myosin-X is sufficient to induce premature compaction. Our findings establish a role for filopodia during preimplantation embryonic development and provide an in vivo context to investigate the biological functions of filopodia in mammals. It has been unclear how round cells elongate during mouse embryo compaction. Plachta and colleagues use live imaging to demonstrate that E-cadherin-dependent filopodia extend to neighbouring cells to drive elongation and compaction.

Nat. Cell Biol. 13, 117–123 (2011); published online 23 January 2011; corrected after print 28 January 2011; In the version of this article initially published online and in print, the values for kout and kin in table 1 were incorrect. The correct values are (×10− 3 s−1). This error has been corrected in both the HTML and PDF versions of the article.

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen’s node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle–related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.