Diverse Routes toward Early Somites in the Mouse Embryo.

Somite formation is foundational to creating the vertebrate segmental body plan. Here, we describe three transcriptional trajectories toward somite formation in the early mouse embryo. Precursors of the anterior-most somites ingress through the primitive streak before E7 and migrate anteriorly by E7.5, while a second wave of more posterior somites develops in the vicinity of the streak. Finally, neuromesodermal progenitors (NMPs) are set aside for subsequent trunk somitogenesis. Single-cell profiling of T-/- chimeric embryos shows that the anterior somites develop in the absence of T and suggests a cell-autonomous function of T as a gatekeeper between paraxial mesoderm production and the building of the NMP pool. Moreover, we identify putative regulators of early T-independent somites and challenge the T-Sox2 cross-antagonism model in early NMPs. Our study highlights the concept of molecular flexibility during early cell-type specification, with broad relevance for pluripotent stem cell differentiation and disease modeling.Research in the authors’ laboratories is supported by the Wellcome Trust, MRC, CRUK, Blood Cancer UK, NIH-NIDDK, the Sanger-EBI Single Cell Centre; by core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute; and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. J.A.G. was funded by Wellcome Trust award [109081/Z/15/A]. C.G. was funded by the Swedish Research Council (2017-06278). This work was funded as part of a Wellcome Strategic Award to study cell fate decisions during gastrulation (105031/D/14/Z) awarded to Wolf Reik, Berthold Göttgens, John Marioni, Jennifer Nichols, Ludovic Vallier, Shankar Srinivas, Benjamin Simons, Sarah Teichmann, and Thierry Voet

Coordinated changes in gene expression kinetics underlie both mouse and human erythroid maturation

Abstract: Background: Single-cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single-cell RNA-Seq permits prediction of future expression states. Here we apply this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis. Results: Intriguingly, RNA velocity correctly identifies epiblast cells as the starting point, but several trajectory predictions at later stages are inconsistent with both real-time ordering and existing knowledge. The most striking discrepancy concerns red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes reveals a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be upregulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1-chimera dataset reveals induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. Conclusions: By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes

GABA promotes gastrin-releasing peptide secretion in NE/NE-like cells: Contribution to prostate cancer progression.

In prostate cancer (PCa), neuroendocrine cells (NE) have been associated with the progression of the disease due to the secretion of neuropeptides that are capable of diffusing and influence surrounding cells. The GABAergic system is enriched in NE-like cells, and contributes to PCa progression. Additionally, γ-aminobutyric acid (GABA) stimulates the secretion of gastrin-releasing peptide (GRP) in peripheral organs. For the first time, in this study we show the role of GABA and GABAB receptor 1 (GABBR1) expression in GRP secretion in NE-like prostate cancer cells. We demonstrated an increase in GRP levels in NE-like cell medium treated with GABAB receptor agonist. Moreover, the blocking of this receptor inhibited GABA-induced GRP secretion. The invasive potential of PC3 cells was enhanced by either GRP or conditioned medium of NE-like cells treated with GABA. Additionally, we confirmed a positive correlation between GABA and GRP levels in the serum of PCa patients with NE markers. Finally, using public available data sets, we found a negative correlation between GABBR1 and androgen receptor (AR) expression, as well as a strong positive correlation between GABBR1 and enolase 2. These results suggest that GABA via GABBR1 induces GRP secretion in NE like cells involved in PCa progression

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.We would like to acknowledge Michal Maj and Line Ericsen, and Kevin Clark in the flow cytometry facilities at the Dunn School and WIMM respectively for providing cell sorting services. The WIMM facility is supported by the MRC HIU; MRC MHU (MC_UU_12009); NIHR Oxford BRC and John Fell Fund (131/030 and 101/517), the EPA fund (CF182 and CF170) and by the WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We thank Neil Ashley for his help on 10x sample preparation and sequencing. The WIMM Single Cell Core Facility was supported by the MRC MHU (MC_UU_12009), the Oxford Single Cell Biology Consortium (MR/M00919X/1) and the WT ISSF (097813/Z/11/B#) funding. The facility was supported by WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We also thank the High-Throughput Genomics Group (Wellcome Trust (WT) Centre for Human Genetics, funded by WT 090532/Z/09/Z), for generating sequencing data. We thank Valerie Kouskoff for providing the iRunx1 ES cell line, Supat Thongjuea and Guanlin Wang for advice with the scRNA-Seq analysis, Joey Riepsaame for advice with CRISP-R experiments, and Doug Higgs, Hedia Chagraoui, Dominic Owens, Andrew Nelson and Arne Mould for helpful discussions. M.D.B and C.P are supported by programmes in the MRC Molecular Hematology Unit Core award (Grant number: MC_UU_12009/2 M.D.B. and MC_UU_12009/9 C.P.). L.G. was supported by a Clarendon PhD studentship and the MRC Molecular Haematology Unit. The work was supported by grants from the Wellcome Trust (214175/Z/18/Z E.J.R, 10281/Z/13/Z L.T.G.H). L.T.G.H was supported by a Clarendon Fund Scholarship and Trinity College Titley Scholarship. E.J.R. is a Wellcome Trust Principal Fellow

Tracking early mammalian organogenesis – prediction and validation of differentiation trajectories at whole organism scale

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.CRUK, Wellcome Trust, MRC, BBSRC, EMBL, E

Computational reconstruction of mouse development using single cell transcriptomics

Single cell transcriptomics has significantly contributed to our understanding of cell types across species, organs and developmental processes. The rapid development of technologies, protocols and computational methods reflects a highly dynamic field over constant improvement. Particularly for Developmental Biology, being able to study at single-cell level and over time is required for a more detailed understanding of the underlying molecular mechanisms associated with cell differentiation and fate choice. Longitudinal cohort studies, ideal for capturing the dynamic nature of developmental mechanisms delineate the temporal relationship between cell type diversity and developmental dynamics. The efforts to build cell atlases from different model organisms already include extensive transcriptomic profiling of embryonic development, where detailed non-human models are essential to compensate the limitations arising from ethical reasons. In this context, the transcriptomic landscape of mouse development is perhaps the most complete among mammals. However, as cells are destroyed when measuring their transcriptomic profiles, only snapshots of the dynamical system are effectively captured. Thus, computational reconstructions of cell differentiation trajectories are essential even when lineage tracing experiments can be performed. The work presented in this dissertation uses a time course large-scale single-cell transcriptomic experiment of mouse embryos to present an effective mathematical and computational strategy that adequately describes the dynamics of gastrulation and early organogenesis on a variety of cell types during mouse embryonic development. The experimental data generated for this project, incorporates new time points into the mouse gastrulation and early organogenesis atlas already publicly available (Pijuan-Sala et al., 2018). That is, an extended version of the existing atlas. Results show a pipeline of sophisticated computational strategies to integrate the new time points. Then, to overcome the challenge of identifying and reconstructing the dynamics of all cell lineages, previously generated knowledge was combined with a variety of state-of-the-art computational methodologies. The analysis of differentiation trajectories is then taken to a deeper level in regards to the emergence of haemato-endothelial lineages, with emphasis on resolving the so-called Primitive and Definitive waves of blood production. Furthermore, perturbations to the system using mouse embryonic chimaera KO models are included. The analysis of haemato-endothelial lineages, presents a detailed reconstruction of the in vivo developmental process not reached by previous studies with single cell transcripomics. Lastly, predicted cell fates are compared to experimental observations by leveraging lineage trace experiments using cell grafting. In summary, this work highlights the complexity associated to generating developmental atlases, how to overcome the corresponding computational challenges and leverage this resource in a variety of contexts

Genetic Diversity of Tick-Borne Rickettsial Pathogens; Insights Gained from Distant Strains

The ability to capture genetic variation with unprecedented resolution improves our understanding of bacterial populations and their ability to cause disease. The goal of the pathogenomics era is to define genetic diversity that results in disease. Despite the economic losses caused by vector-borne bacteria in the Order Rickettsiales, little is known about the genetic variants responsible for observed phenotypes. The tick-transmitted rickettsial pathogen Anaplasma marginale infects cattle in tropical and subtropical regions worldwide, including Australia. Genomic analysis of North American A. marginale strains reveals a closed core genome defined by high levels of Single Nucleotide Polymorphisms (SNPs). Here we report the first genome sequences and comparative analysis for Australian strains that differ in virulence and transmissibility. A list of genetic differences that segregate with phenotype was evaluated for the ability to distinguish the attenuated strain from virulent field strains. Phylogenetic analyses of the Australian strains revealed a marked evolutionary distance from all previously sequenced strains. SNP analysis showed a strikingly reduced genetic diversity between these strains, with the smallest number of SNPs detected between any two A. marginale strains. The low diversity between these phenotypically distinct bacteria presents a unique opportunity to identify the genetic determinants of virulence and transmission