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We also included natural variation in sulfur concentrations by offering Loripes collected at two different locations. In both cases lower sulfur levels in Loripes resulted in higher consumption rates in red knots. Over time the red knots increased their intake rates on Loripes, showing their ability to adjust to a higher intake of sulfur.The nuclear envelope (NE) separates genomic DNA from the cytoplasm and provides the molecular platforms for nucleocytoplasmic transport, higher-order chromatin organization, and physical links between the nucleus and cytoskeleton. Recent studies have shown that the NE is often damaged by various stresses termed "NE stress", leading to critical cellular dysfunction. Accumulating evidence has revealed the crucial roles of NE stress in the pathology of a broad spectrum of diseases. In the central nervous system (CNS), NE dysfunction impairs neural development and is associated with several neurological disorders, such as Alzheimer's disease and autosomal dominant leukodystrophy. In this review, the structure and functions of the NE are summarized, and the concepts of NE stress and NE stress responses are introduced. Additionally, the significant roles of the NE in the development of CNS and the mechanistic connections between NE stress and neurological disorders are described.An indispensable prerequisite of mammalian development is successful morphogenesis in the epiblast, the embryonic tissue that gives rise to all differentiated cells of the adult mammal. The right control of both epiblast morphogenesis and the events that regulate its shape in particular during implantation is henceforth of tremendous importance. However, monitoring the process of development in implanting human embryos is ethically and technically challenging, making it difficult to troubleshoot when things go wrong, as it is unfortunately the case with over 30% of pregnancy failures. Although modern in vitro techniques have proven very insightful lately, more tools are needed in the quest to elucidate mammalian and human development. Mathematical and computational modeling position themselves as helpful complementary tools in the biologist's toolbox, enabling the exploration of the living in silico, beyond the boundaries set by ethical concerns and the potential limitations of wet lab techniques. Here, we show how mathematical modeling and computer simulations can be used to emulate and investigate mechanisms driving epiblast shape changes in mouse and human embryos during implantation.Here we describe a method to engraft epiblast stem cells (EpiSC) into the epiblast of gastrulation-stage mouse embryo to test the lineage propensity acquired by the EpiSCs during in vitro culture under different signaling conditions. After dissection and grafting, the recipient embryos can be grown in whole-embryo culture for up to 48 h and the contribution of the EpiSC-derived cells to tissues in the recipient embryo is assessed by light sheet 3D microscopy.During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. Progress on these issues is slowed by the difficulty to record or perturb the patterning events in real time, especially in mammalian embryos that develop in utero. Because they permit easy observation and manipulation, in vitro model systems offer an exciting opportunity to dissect the rules governing the organization of the mammalian gastrula. For instance, it is sufficient to cultivate human embryonic stem cells on micropatterned substrates to reveal their self-organization potential. We present here a method to obtain micropatterned mouse Epiblast Like Cells colonies, providing a convenient way to compare spatial organization of mouse and human pluripotent stem cells and to complement the characterization of mutant embryos in a controlled environment.In humans, germ cells are specified in the extraembryonic yolk sac, at proximity of allantois, around the second week of gestation. Derivation of human germ cell-like cells (hPGCLCs) from human pluripotent cells in vitro is of a great importance for research purposes, such as disease modeling, or studying the early human germ cell development and the effect of environmental factors on this development. As it is not possible to access human embryos at early developmental stages, a two-step protocol has been proposed by Sasaki and colleagues to differentiate hPGCLCs in vitro from human pluripotent stem cells. Here, we report a detailed protocol for in vitro hPGCLCs differentiation from induced pluripotent stem cells (iPSCs).The ability to generate primordial germ cell-like cells (PGCLCs) from murine embryonic stem cells (ESCs) has enabled in vitro investigation of the molecular mechanisms regulating this process without the use of a mouse model. Here we describe the procedures from the culture of ESCs to the detection of PGCLCs in the embryoid bodies (spheroids).This chapter describes the protocol to derive definitive endoderm cells from epiblast stem cells (EpiSCs) via a process analogous to gastrulation in embryos. The basis of this protocol mimicking the in vivo gastrulation process makes a contrast with those using sequential administration of pharmacological molecules and recombinant signaling proteins even at nonphysiological levels. In the experimental setup, EpiSCs are first freed from the dish-adherent condition to form free-floating aggregates, where endoderm precursor pools are produced. Embedding the EpiSC aggregates in the Matrigel allows the endoderm precursors to interact with the Matrigel mimicking the laminin-rich basement membrane underlying the egg cylinder epiblast in embryos, and let the precursors migrate into the Matrigel-filled external zone and develop into endodermal epithelial tissues.The different states of mouse pluripotency described so far rely on a combination of molecular, phenotypic, and functional analysis. Embryonic Stem cells (ESCs) aggregated in suspension culture are able to form 3D embryo-like structures called gastruloids that mimic features of the gastrulation process. Recent findings indicate that gastruloid formation efficiency decreases as pluripotency progresses from naïve to primed state, and suggest that gastruloids formation may represent a functional assay to discriminate different states of mouse pluripotency.Here we describe a method to generate gastruloids from Epiblast-like cells (EpiLCs), which are transiently induced from ESCs by Activin A and bFGF and represent an intermediate state from naïve ESCs to primed Epiblast Stem cells.Glycosylation is one of the most abundant posttranslational modifications and is involved in a wide range of cellular processes. Glycome diversity in mammals is generated by the action of over 200 distinct glycosyltransferases and related enzymes. Nevertheless, glycosylation dynamics are tightly coordinated to allow proper organismal development. Here, using mouse embryonic stem cells (mESCs) and mouse epiblast-like cells (mEpiLCs) as model systems, we describe a robust protocol that allows comprehensive and comparative structural analysis of the glycome.In this methods chapter, we describe the use of isobaric tags for relative and absolute quantification (iTRAQ) for the differential expression analysis of global proteins between embryonic stem cell samples. This protocol describes how proteins are collected from cell culture, digested and prepared so that peptides are labeled with these isobaric tags. SRT2104 Labeled digests are pooled, fractionated offline, and quantified using liquid chromatography-mass spectrometry (LC-MS). This offline fractionation allows for a greater separation and thus increased identification/quantification of peptides. This combined method enables large-scale, deep penetration into the proteome of embryonic stem cells. During quantification, the relative intensities of label-derived reporter ions represent the relative amount of peptide in each sample. Using search algorithms that integrate the generated data for the identified and quantified peptides allows the relative quantification of proteins in the samples. The isobaric tags can be used in a 4 or 8 multiplexed manner; however, using an 8-plex experimental setup allows for the simultaneous analysis of biological and technical replicates within the same mass spectrometry run, thus minimizing experimental variation and increasing the confidence in any identified expression differences.Single-cell transcriptome analysis reveals heterogeneous cell types in complex tissues and leads to unexpected biological findings when compared to bulk populations. However most of the methods focus on the 3'-end of polyadenylated transcripts using droplet-based technology. To achieve complete transcriptome, we describe single-cell 5'-end transcriptome protocol with random primed-cDNA harvesting on the Fluidigm C1™ platform which can isolate and process up to 96 cells from a single run with custom library preparation. The method enables detection of Transcription Start Site (TSS) at the single-cell resolution yielding a more comprehensive overview of gene regulatory elements governing in the EpiSC-like cell (EpiLC) including non-polyadenylated RNA and enhancer RNA activities.Single-cell RNA-sequencing (scRNA-Seq) is a widely used technology to reveal the heterogeneity and dynamics of tissues, organisms, and complex diseases. Here, a workflow is presented for preprocessing of scRNA-seq data to quantify gene abundances in individual cells followed by visualization and annotation of cells.The assay for transposase-accessible chromatin using sequencing (ATAC-seq) is used to identify open chromatin regions in cells. This can be used to identify putative regulatory regions, determine dynamics and mechanisms of transcription factors when coupled with ChIP-seq and predict interactions between proteins and chromatin. Compared to previous methods, MNase-seq and DNase-seq, ATAC-seq requires only 50,000 cells, orders of magnitude fewer cells. In addition, the ATAC-seq protocol takes one day to progress from cells to sequencing ready libraries.Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.