The embryonic node behaves as an instructive stem cell niche for axial elongation

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

Bridging the N-terminal and middle domains in FliG of the flagellar rotor

Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function

Integrated single-cell RNA sequencing analysis reveals alterations of ageing human lung endothelium heterogeneity in idiopathic pulmonary fibrosis

Increasing age is the main risk factor for chronic lung diseases (CLD) including idiopathic pulmonary fibrosis (IPF). Halting or reversing progression of IPF remains an unmet clinical need due to limited knowledge of underlying mechanisms. In particular, the contribution of the endothelium to ageing in human lung under physiological conditions and in IPF remains insufficiently understood. In this study, we analysed heterogeneity of endothelium in physiologically ageing human lung and its alterations in IPF. We conducted a comprehensive in silico analysis of scRNAseq profiles of human lung tissues from older healthy donors and age-matched IPF patients (n=9 for each group) by integrating datasets from two independent cohorts. We generated a single-cell map of the ageing human lung and identified 17 subpopulations of ageing endothelium (12 for blood and 5 for lymphatic vessels, including 4 “de-differentiated”), with distinct transcriptional profiles, specific gene expression signatures and percentage contributions, revealing previously underappreciated extent of heterogeneity. In IPF lung, the balance of different endothelial sub-types was significantly altered both in terms of cell numbers and gene expression patterns, identifying disease-relevant subpopulations and transcriptional changes associated with specific signalling pathways and cellular processes. These findings reveal a previously unrecognised phenomenon of ageing human lung endothelium re-programming towards an “IPF endothelium” state, suggesting potential avenues for therapeutic management or biomarker discovery for diagnostics or prognostics of IPF. Our study creates a conceptual framework for appreciating the heterogeneity of ageing endothelium and its alterations in CLDs and diseases associated with fibrosis in other organs, including lymphoedema and cancer

Sequential changes in cellular properties accompanying amniote somite formation

Somites are transient structures derived from the pre-somitic mesoderm (PSM), involving mesenchyme-to-epithelial transition (MET) where the cells change their shape and polarize. Using Scanning electron microscopy (SEM), immunocytochemistry and confocal microscopy, we study the progression of these events along the tail-to-head axis of the embryo, which mirrors the progression of somitogenesis (younger cells located more caudally). SEM revealed that PSM epithelialization is a gradual process, which begins much earlier than previously thought, starting with the dorsalmost cells, then the medial ones, and then, simultaneously, the ventral and lateral cells, before a somite fully separates from the PSM. The core (internal) cells of the PSM and somites never epithelialize, which suggests that the core cells could be ‘trapped’ within the somitocoele after cells at the surfaces of the PSM undergo MET. Three-dimensional imaging of the distribution of the cell polarity markers PKCζ, PAR3, ZO1, the Golgi marker GM130 and the apical marker N-cadherin reveal that the pattern of polarization is distinctive for each marker and for each surface of the PSM, but the order of these events is not the same as the progression of cell elongation. These observations challenge some assumptions underlying existing models of somite formation

The nuclear lamina couples mechanical forces to cell fate in the preimplantation embryo via actin organization

Abstract 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

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity.

Acknowledgements: We are grateful to Sandip Patel, Anant Parekh, Andrea Streit and Octavian Voiculescu for advice on experiments and helpful comments on the manuscript. We also acknowledge the Cell Services Science Technology Platform at the Francis Crick Institute for providing regular Mycoplasma screening of the hESC line, the Zoology Imaging Facility of Cambridge University for assistance and support with microscopy on Drosophila embryos, and Sophie Brumm for providing a pSMAD staining protocol. This study was funded by a Wellcome Trust Investigator Award (107055/Z/15/Z) to C.D.S., which supported H.C.L., H.-C.L. and N.M.M.O. H.C.L. was also supported by a fellowship from the Basic Science Research Program through the National Research Foundation of Korea (NRF) (2014R1A6A3A03053468). N.M.M.O. was also funded by UCL. C.H. was funded by a Medical Research Council Doctoral Training Programme studentship (MR/N013867/1). P.B.-B. and N.M. were funded by the Francis Crick Institute which receives its core funding from Cancer Research UK (CC2186), the UK Medical Research Council (CC2186), and the Wellcome Trust (CC2186). A.A.M. was funded by a studentship from the Anatomical Society of Great Britain and Ireland in C.D.S.’s lab. Y.Z. and J.F. were funded by the Michigan-Cambridge Research Initiative, the US National Institutes of Health (R21 HD100931), and the National Science Foundation (CMMI 1917304). E.L.W. was funded by a BBSRC DTP scholarship. T.T.W. was funded by the University of Cambridge ISSF (097814) and the Wellcome Trust (200734/Z/16/Z).Funder: Anatomical Society of Great Britain and Ireland. PhD studentshipFunder: Michigan-Cambridge Research InitiativeFunder: U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity

Abstract Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity.

Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin