Cell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity; however, existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin‐associated factors that modulate levels of transcriptional noise. Herein, we show destabilization of pluripotency‐associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl‐transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5), have been inhibited. Functionally, network destabilization associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we show evidence of a relationship between transcriptional heterogeneity and cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells, suggesting a general principle that could be exploited in other normal and malignant stem cell fate transitions. stem cells 2018;36:1828–11

Gene expression is a fundamental cellular process by which proteins are eventually synthesized based on the information coded in the genes. This process includes four major steps: transcription of the DNA segment corresponding to a gene to mRNA molecules, the degradation of the mRNA molecules, the translation of mRNA molecules to proteins by the ribosome and the degradation of the proteins. We present an innovative quantitative study of the interaction between the gene translation stage and the mRNA degradation stage using large scale genomic data of S. cerevisiae, which include measurements of mRNA levels, mRNA half-lives, ribosomal densities and protein abundances, for thousands of genes. The reported results support the conjecture that transcripts with higher ribosomal density, which is related to the translation stage, tend to have elevated half-lives, and we suggest a novel quantitative estimation of the strength of this relation. Specifically, we show that on average, an increase of 78% in ribosomal density yields an increase of 25% in mRNA half-life, and that this relation between ribosomal density and mRNA half-life is not function specific. In addition, our analyses demonstrate that ribosomal density along the entire ORF, and not in specific locations, has a significant effect on the transcript half-life. Finally, we show that the reported relation cannot be explained by different expression levels among genes. A plausible explanation for the reported results is that ribosomes tend to protect the mRNA molecules from the exosome complexes degrading them; however, additional non-mutually exclusive possible explanations for the reported relation and experiments for their verifications are discussed in the paper.

The mammalian embryo's caudal lateral epiblast (CLE) harbours bipotent progenitors, called neural mesodermal progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here, we performed a single cell analysis of different in vitro NMP populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them with E8.25 CLE mouse embryos. In our analysis of this region, our findings challenge the notion that NMPs can be defined by the exclusive co-expression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built support vector machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which suggests that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMP population.

The caudal lateral epiblast of mammalian embryos harbours bipotent progenitors that contribute to the spinal cord and the paraxial mesoderm in concert with the body axis elongation. These progenitors, called neural mesodermal progenitors (NMPs), are identified as cells that co-express Sox2 and T/brachyury, a criterion used to derive NMP-like cells from embryonic stem cells in vitro. However, unlike embryonic NMPs, these progenitors do not selfrenew. Here, we find that the protocols that yield NMP-like cells in vitro initially produce a multipotent population that, in addition to NMPs, generates progenitors for the lateral plate and intermediate mesoderm. We show that epiblast stem cells (EpiSCs) are an effective source of these multipotent progenitors, which are further differentiated by a balance between BMP and Nodal signalling. Importantly, we show that NMP-like cells derived from EpiSCs exhibit limited self-renewal in vitro and a gene expression signature like their embryonic counterparts. RESULTSEpiSCs yield a postimplantation epiblast population that resembles the CLE Several protocols allow the differentiation of ESCs into an NMPlike population, defined as cells that co-express T and Sox2 that can be further differentiated into neural and mesodermal progenitors (summarized by Henrique et al., 2015). However, it is not clear

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-β signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

During vertebrate embryogenesis, all mesodermal and ectodermal derivatives posterior to the hindbrain, derived from a pool of progenitors, localized at the caudal end of the embryo, that are maintained in an uncommitted state as the body axis elongates. In mammals, these progenitors are located in the Caudal Lateral Epiblast (CLE), behind the node at the posterior end of the embryo, and include a stem cell/progenitor zone of bipotent progenitors that contribute to both the elongating spinal cord and the paraxial mesoderm. These progenitors, called the Neural Mesodermal Progenitors (NMPs), are often identified by the coexpression of Sox2 and T. Using this criterion for identification, a number of in vitro studies have been able to produce NMP-like cells from Pluripotent Stem Cells (PSCs) but, in contrast with the embryonic NMPs, these in vitro counterparts do not self renew and exhibit limited ability to contribute to the lengthening tissues in allo or xenotransplant assays. Here we use different protocols for NMP-like cells and find that, in addition to NMPs, all produce cells with the potential to differentiate into Lateral Plate and Intermediate Mesoderm precursors. We also show that Epiblast Stem Cells (EpiSCs) are a better starting source to produce these multipotent progenitors and NMPs than ESCs and this manifests in three aspects: introducing these cells to a specific differentiation protocol yields cells with gene expression patterns with a characteristic of NMPs; an ability to self renew in vitro and to contribute progressively to both neural and mesodermal compartments along more posterior portions of the embryonic body axis in a xenotransplant assay. These NMP-like cells are derived from a population that resembles the Caudal Epiblast (CE) of the embryo at the time of the appearance of the node. Our study also shows how a balance between BMP and Nodal signalling is likely to influence the patterning of this multipotent population suggesting that at the end of gastrulation and associated with the node, a multipotential progenitor population emerges that will give rise to the spinal cord and the mesodermal populations posterior to the brain.

Gene expression is a fundamental cellular process by which proteins are synthesized based on the information coded in the genes. The two major steps of this process are the transcription of the DNA segment corresponding to a gene to mRNA molecules and the translation of the mRNA molecules to proteins by the ribosome. Thus, understanding, modeling and engineering the different stages of this process have both important biotechnological applications and contributions to basic life science. In previous studies we have introduced the Homogenous Ribosome Flow Model (HRFM) and demonstrated its advantages in analyses of the translation process. In this study we introduce the RNA Polymerase Flow Model (RPFM), a non trivial extension of the HRFM, which also includes a backward flow and can be used for modeling transcription and maybe other similar processes. We compare the HRFM and the RPFM in the three regimes of the transcription process: rate limiting initiation, rate limiting elongation and rate limiting termination via a simulative and analytical analysis. In addition, based on experimental data, we show that RPFM is a better choice for modeling transcription process.

Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal, endothelial) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, while the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirement for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as Pancreas Embryonic Cell Atlas).