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Oikonomou, P.

Publications and source records attributed to Oikonomou, P..

11 recordsLinked to original sources

An endodermal subpopulation generates neural and mesodermal fates in the posterior chick embryo

Embryogenesis occurs through a progressive narrowing of cell fate potential, initiating with the segregation of three distinct germ layers during gastrulation. Although classically, each germ layer contributes to distinct tissue types as development proceeds, this view has been revised with the discovery of neuromesodermal progenitors (NMPs) -- a bipotent progenitor population in the posterior embryo that gives rise to traditionally ectodermal and mesodermal tissues after gastrulation has concluded. However, until now the notion of lineage restriction of the endoderm to gastrointestinal, respiratory, and endocrine tissues has largely remained intact. Here, we describe a unique subpopulation in the chick endoderm that initially lines the ventral surface of Hensens node (the amniote organizer). As posterior regression of the node ends with termination of the primitive streak, these cells undergo an FGF-dependent epithelial-to-mesenchymal transition, erasing their endodermal identity as they invade the tailbud and subsequently differentiate into a remarkably broad range of cell types including paraxial, lateral plate, and intermediate mesoderm, and to a lesser extent, notochord and neural tube. Disrupting ingression of node endoderm reduced embryonic axis elongation -- a process attributed to mesoderm -- by 50%. Through lineage barcoding, single-cell RNA sequencing, and fate mapping experiments, we conclude that the endodermal compartment of Hensens node harbors a mixed population of fate restricted and multipotent progenitor cells that give rise to clonal populations spanning traditional germ layer boundaries. These findings illustrate a surprising example of germ layer plasticity and fate convergence across distant progenitor populations during amniote development.

developmental biology↗

Adaptation by stochastic tuning of gene expression in mammalian cells

Cells often face environmental challenges for which they lack pre-programmed regulatory responses. We have previously shown that yeast cells can, nevertheless, adapt to such unfamiliar conditions by a phenomenon we have termed stochastic tuning. In this process, cells use gene expression noise to randomly change the expression of individual genes and actively reinforce those changes that improve the overall health of the cell. Here, we provide experimental evidence for stochastic tuning in human cells adapting to the lethal chemotherapeutic agent methotrexate. As in yeast, the tuning process is not driven by mutations and is reversible upon removal of methotrexate challenge. We show that stochastic tuning is a conserved eukaryotic mechanism of cellular adaptation, and potentially a key factor in the phenomenon of cancer cell plasticity underlying chemotherapy resistance.

systems biology↗

Convergent flow-mediated mesenchymal force drives embryonic foregut constriction and splitting

The transformation of a two-dimensional epithelial sheet into various three-dimensional structures is a critical process in generating the diversity of animal forms. Previous studies of epithelial folding have revealed diverse mechanisms driven by epithelium-intrinsic or -extrinsic forces. Yet little is known about the biomechanical basis of epithelial splitting, which involves extreme folding and eventually a topological transition breaking the epithelial tube. Here, we leverage tracheal-esophageal separation (TES), a critical and highly conserved morphogenetic event during tetrapod embryogenesis, as a model system for interrogating epithelial tube splitting both in vivo and ex vivo. Comparing TES in chick and mouse embryos, we identified an evolutionarily conserved, compressive force exerted by the mesenchyme surrounding the epithelium, as being necessary to drive epithelial constriction and splitting. The compressive force is mediated by localized convergent flow of mesenchymal cells towards the epithelium. We further found that Sonic hedgehog (SHH) secreted by the epithelium functions as an attractive cue for mesenchymal cells. Removal of the mesenchyme, inhibition of cell migration, or loss of SHH signaling all abrogate TES, which can be rescued by externally applied pressure. These results unveil the biomechanical basis of epithelial splitting and suggest plausible mesenchymal origins of tracheal-esophageal birth defects.

developmental biology↗

The genetic landscape of antibiotic sensitivity in Staphylococcus aureus

A comprehensive genetic landscape of antibiotic sensitivity in Staphylococcus aureus is lacking. Using ultra-dense CRISPR-interference libraries, we systematically quantified global gene fitness across ten antibiotics and uncovered hundreds of significant antibiotic-gene interactions. Essential genes dominated these interactions, a finding not revealed by transposon-based studies. Processes most vulnerable to transcriptional repression under antibiotic conditions included cell wall synthesis/cell division (CC), DNA replication/DNA recombination (DD), coenzyme A biosynthesis, and riboflavin metabolism. Network and genetic analyses further revealed novel synergistic genetic interactions (GIs) within these processes, including an extensive CC-DD subnetwork. Only a subset of CC-DD synergies was dependent on the cell division inhibitor SosA. Informed by these GIs, we identified multiple drug-drug combinations with potent synergistic activity against multidrug-resistant S. aureus. Our detailed profiling of drug-gene, gene-gene, and drug-drug interactions reveals novel functional relationships among essential genes and defines a vulnerability landscape to guide new drug target discovery and effective combination therapies.

systems biology↗

Material Properties Of The Embryonic Small Intestine During Buckling Morphogenesis

During embryonic development, tissues undergo dramatic deformations as functional morphologies are stereotypically sculpted from simple rudiments. Formation of healthy, functional organs therefore requires tight control over the material properties of embryonic tissues during development, yet the biological basis of embryonic tissue mechanics is poorly understood. The present study investigates the mechanics of the embryonic small intestine, a tissue that is compactly organized in the body cavity by a mechanical instability during development, wherein differential elongation rates between the intestinal tube and its attached mesentery create compressive forces that buckle the tube into loops with wavelength and curvature that are tightly conserved for a given species. Focusing on the intestinal tube, we combined micromechanical testing with histologic analyses and enzymatic degradation experiments to conclude that elastic fibers closely associated with intestinal smooth muscle layers are responsible for the bending stiffness of the tube, and for establishing its pronounced mechanical anisotropy. These findings provide insights into the developmental role of elastic fibers in controlling tissue stiffness, and raise new questions on the physiologic function of elastic fibers in the intestine during adulthood. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/606927v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1bbf9d9org.highwire.dtl.DTLVardef@10428eorg.highwire.dtl.DTLVardef@181b530org.highwire.dtl.DTLVardef@9e758c_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

Application of tissue-scale tension to avian epithelia in vivo to study multiscale mechanical properties and inter-germ layer coupling

As cross-disciplinary approaches drawing from physics and mechanics have increasingly influenced our understanding of morphogenesis, the tools available to measure and perturb physical aspects of embryonic development have expanded as well. However, it remains a challenge to measure mechanical properties and apply exogenous tissue-scale forces in vivo, particularly for epithelia. Exploiting the size and accessibility of the developing chick embryo, here we describe a simple technique to quantitatively apply exogenous forces on the order of 1-100 N to the endodermal epithelium. To demonstrate the utility of this approach, we performed a series of proof-of-concept experiments that reveal fundamental and unexpected mechanical behaviors in the early chick embryo, including mechanotype heterogeneity among cells of the midgut endoderm, complex non-cell autonomous effects of actin disruption, and a high degree of mechanical coupling between the endoderm and adjacent paraxial mesoderm. To illustrate the broader utility of this method, we determined that forces on the order of 10 N are sufficient to unzip the neural tube during primary neurulation. Together, these findings provide basic insights into the mechanics of embryonic epithelia in vivo in the early avian embryo, and provide a useful tool for future investigations of how morphogenesis is influenced by mechanical factors. Graphical AbstractO_ST_ABSSummary StatementC_ST_ABS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/588089v2_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@7f0d90org.highwire.dtl.DTLVardef@153a4b0org.highwire.dtl.DTLVardef@614051org.highwire.dtl.DTLVardef@c5b432_HPS_FORMAT_FIGEXP M_FIG C_FIG A simple approach is devised to quantitatively apply tension to epithelia in vivo, and used to study endoderm mechanics in the deeloping chick embryo.

developmental biology↗

A chemo-mechanical model of endoderm movements driving elongation of the amniote hindgut

While mechanical and biochemical descriptions of development are each essential, integration of upstream morphogenic cues with downstream tissue mechanics remains understudied in many contexts during vertebrate morphogenesis. A posterior gradient of Fibroblast Growth Factor (FGF) ligands generates a contractile force gradient in the definitive endoderm, driving collective cell movements to form the hindgut. Here, we developed a two-dimensional chemo-mechanical model to investigate how mechanical properties of the endoderm and transport properties of FGF coordinately regulate this process. We began by formulating a 2-D reaction-diffusion-advection model that describes the formation of an FGF protein gradient due to posterior displacement of cells transcribing unstable Fgf8 mRNA during axis elongation, coupled with translation, diffusion, and degradation of FGF protein. This was used together with experimental measurements of FGF activity in the chick endoderm to inform a continuum model of definitive endoderm as an active viscous fluid that generates contractile stresses in proportion to FGF concentration. The model replicated key aspects of hindgut morphogenesis, confirms that heterogeneous - but isotropic - contraction is sufficient to generate large anisotropic cell movements, and provides new insight into how chemomechanical coupling across the mesoderm and endoderm coordinates hindgut elongation with outgrowth of the tailbud. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC="FIGDIR/small/541363v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@abb6fborg.highwire.dtl.DTLVardef@17a297eorg.highwire.dtl.DTLVardef@13a454borg.highwire.dtl.DTLVardef@9b699d_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary statementThis study employs a mathematical model to investigate the interplay between morphogen gradients and tissue mechanics in regulating the collective cell movements that drive hindgut morphogenesis in the chick embryo.

developmental biology↗

Molecular topography of an entire nervous system

Nervous systems are constructed from a deep repertoire of neuron types but the underlying gene expression programs that specify individual neuron identities are poorly understood. To address this deficit, we have produced an expression profile of all 302 neurons of the C. elegans nervous system that matches the single cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses unique codes of [~]23 neuropeptide-encoding genes and [~]36 neuropeptide receptors thus pointing to an expansive "wireless" signaling network. To demonstrate the utility of this uniquely comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression across the nervous system and (2) reveal adhesion proteins with potential roles in synaptic specificity and process placement. These data are available at cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity and function throughout the C. elegans nervous system.

neuroscience↗

An inducible CRISPR-interference library for genetic interrogation of Saccharomyces cerevisiae biology

Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite its dominance as a model eukaryote, a genome-wide CRISPRi library, optimized for targeting the Saccharomyces cerevisiae genome, has not been presented to date. We have generated a comprehensive, inducible CRISPRi library, based on spacer design rules optimized for yeast. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed parameters for optimal transcriptional repression, including upstream distance, nucleosomal occupancy, and strand bias. CRISPRi screens, using this library can identify genes and pathways with high precision and low false discovery rate across a variety of experimental conditions, enabling rapid and reliable genome-wide assessment of gene function and genetic interactions in S.cerevisiae.

systems biology↗

Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

Despite longstanding appreciation of gene expression heterogeneity in isogenic bacterial populations, affordable and scalable technologies for studying single bacterial cells have been limited. While single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems, application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance, lack of mRNA polyadenylation, and thick cell walls. Here, we present Prokaryotic Expression-profiling by Tagging RNA In Situ and sequencing (PETRI-seq), a low-cost, high-throughput, prokaryotic scRNA-seq pipeline that overcomes these technical obstacles. PETRI-seq uses in situ combinatorial indexing to barcode transcripts from tens of thousands of cells in a single experiment. PETRI-seq captures single cell transcriptomes of Gram-negative and Gram-positive bacteria with high purity and low bias, with median capture rates >200 mRNAs/cell for exponentially growing E. coli. These characteristics enable robust discrimination of cell-states corresponding to different phases of growth. When applied to wild-type S. aureus, PETRI-seq revealed a rare sub-population of cells undergoing prophage induction. We anticipate broad utility of PETRI-seq in defining single-cell states and their dynamics in complex microbial communities.

systems biology↗

A post-transcriptional regulatory code for mRNA stability during the zebrafish maternal-to-zygotic transition

Post-transcriptional regulation is crucial to shape gene expression. During the Maternal-to-Zygotic Transition (MZT), thousands of maternal transcripts are regulated upon fertilization and genome activation. Transcript stability can be influenced by cis-elements and trans-factors, but how these inputs are integrated to determine the overall mRNA stability is unclear. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. To identify cis-regulatory elements, we performed a massively parallel reporter assay for stability-influencing sequences, which revealed that 3-UTR poly-U motifs are associated with mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC and CUGC elements emerged as the main destabilizing motifs in the embryo, with miR-430 and AREs causing mRNA deadenylation in a genome activation-dependent manner. To identify the trans-factors interacting with these cis-elements, we comprehensively profiled RNA-protein interactions and their associated regulatory activities across the transcriptome during the MZT. We find that poly-U binding proteins are preferentially associated with 3-UTR sequences and stabilizing motifs, and that antagonistic sequence contexts for poly-C and poly-U binding proteins shape the binding landscape and magnitude of regulation across the transcriptome. Finally, we integrate these regulatory motifs into a machine learning model that accurately predicts the stability of mRNA reporters in vivo. Our findings reveal how mechanisms of post-transcriptional regulation are coordinated to direct changes in mRNA stability within the early zebrafish embryo.

genomics↗