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Results for “developmental biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Control and mechanisms of pulsatile flows in epithelial monolayers

Collective motions of epithelial cells in vivo are essential for morphogenesis in developmental biology. Tissues elongate, contract, flow, and oscillate, thus sculpting embryos. These tissue level dynamics are known, but the physical mechanisms at the cellular level are unclear, with various behaviors depending on the tissues and species. Moreover, investigations on in vitro tissue behavior usually focus on only one type of cell dynamics and use diverse theoretical approaches, making systematic comparisons between studies challenging. Here, we show that a single epithelial monolayer of Madin Darby Canine Kidney (MDCK) cells can exhibit two types of local tissue kinematics, pulsations and long range coherent flows. We analyzed these distinct motions by using quantitative live imaging. We also report that these motions can be controlled with internal and external cues such as specific inhibitors, and friction modulation of the substrate by microcontact printing method. We further demonstrate with a unified vertex model that both behaviors depend on the competition between velocity alignment and random diffusion of cell polarization. When alignment and diffusion are comparable, a pulsatile flow emerges, whereas the tissue undergoes long-range flows when velocity alignment dominates. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics are important in regulating the dynamics of tissue morphogenesis.

biophysics↗

bioRxiv2020

Development and application of aerobic, chemically defined media for Dysgonomonas

Members of Dysgonomonas are Gram-negative, non-motile, facultatively anaerobic coccobacilli originally described in relation to their isolation stool and wounds of human patients (CDC group DF-3). More recently Dysgonomonas have been found to be widely distributed in terrestrial environments and are particularly enriched in insect systems. Their prevalence in xylophagous insects such as termites and wood-feeding cockroaches, as well as in soil-fed microbial fuel cells, elicit interest in lignocellulose degradation and biofuel production, respectively. Their prevalence in mosquito and fruit fly have implications relating to symbiosis, host immunology and developmental biology. Additionally, their prevalence in termite, mosquito and nematode present novel opportunities for pest and vector control. Currently, the absolute growth requirements of Dysgonomonas are unknown, and they are cultured solely under anaerobic conditions on complex media containing blood, peptones, tryptones, and yeast, plant or meat extracts. Restrictive & undefined culturing conditions preclude physiological and genetic studies, and thus further understanding of metabolic potential. Here we describe the requirements for growth of termite-derived Dysgonomonas isolates and create parallel defined, minimal and complex media that permit vigorous and reliable aerobic growth. Furthermore, we show that these media can be used to easily enrich for Dysgonomonas isolates from complex and microbially-diverse environmental samples. Impact StatementMembers of the genus Dysgonomonas are increasingly prevalent in ecological, medical and biotechnological contexts. To the best of our knowledge, there are currently no formulations for chemically defined or minimal media for Dysgonomonas, and limited complex formulations that allow aerobic growth, particularly on solid media. We have created three parallel media formulations (complex, defined & minimal) that permit robust aerobic and anaerobic growth in liquid and agar-solidified media. These formulations remove the necessity for animal blood and expensive equipment such as anaerobic chambers, which can inhibit basic research by groups with biosafety and resource limitations.

microbiology↗

bioRxiv2020

Global Profiling of the Lysine Crotonylome in Different Pluripotent States

Pluripotent stem cells (PSCs) can be expanded in vitro in different culture conditions, resulting in a spectrum of cell states with distinct properties. Understanding how PSCs transition from one state to another, ultimately leading to lineage-specific differentiation, is important for developmental biology and regenerative medicine. Although there is significant information regarding gene expression changes controlling these transitions, less is known about post-translational modifications of proteins. Protein crotonylation is a newly discovered post-translational modification where lysine residues are modified with a crotonyl group. Here, we employed affinity purification of crotonylated peptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to systematically profile protein crotonylation in mouse PSCs in different states including ground, metastable and primed state, as well as metastable PSCs undergoing early pluripotency exit. We successfully identified 3628 high-confidence sites in 1426 proteins. These crotonylated proteins are enriched for factors involved in functions related to pluripotency such as RNA biogenesis, central carbon metabolism and proteasome function. Moreover, we found that increasing the cellular levels of crotonyl-CoA through crotonic acid treatment promotes proteasome activity in metastable PSCs and delays their differentiation, consistent with previous observations showing that enhanced proteasomal activity helps to sustain pluripotency. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of metabolism in other cell fate transitions.

cell biology↗

bioRxiv2020

Reprogramming Epiblast Stem Cells into Pre-Implantation Blastocyst Cell-like Cells

Recently, a new wave of synthetic embryo systems (SESs) have been established from cultured cells toward efficient and ethical embryonic development research. We recently reported our epiblast stem cell (EPISC) reprogramming SES that generates numerous blastocyst (BC)-like hemispheres (BCLH) with pluripotent and extraembryonic cell features detected microscopically. Here, we further explored the system over key time points with unprecedented single-cell RNA sequencing (scRNA-seq) analysis and revealed broad induction of the 2C-like reporter MERVL and RNA velocity diverging three major population regions with genetic expression resembling pluripotent epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). Enrichment of those three BC-like cell fates involved key regulons, zygotic genome activation (ZGA) related genes, specific RNA splicing, and select cells meaningfully distinguished critical regulons of model cells. This analysis confirms the induction of the extraembryonic cell populations during the reprogramming and we anticipate that our unique BCLH SES and rich data may uncover new facets of cell potency, improve developmental biology, and help biomedicine advance.

synthetic biology↗

bioRxiv2020

Streamlined and quantitative detection of chimerism in mouse tissue using digital PCR

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues has challenges. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among albino and non-albino mouse strains. In addition, we have validated that this assay is compatible with crude lysate from most organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Importantly, the concepts developed and discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

genetics↗

bioRxiv2020

Coarse-grained modeling of mitochondrial metabolism enables subcellular flux inference from fluorescence lifetime imaging microscopy

Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the FLIM measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.

biophysics↗

bioRxiv2020

Operating principles of circular toggle polygons

Decoding the dynamics of cellular decision-making and cell differentiation is a central question in cell and developmental biology. A common network motif involved in many cell-fate decisions is a mutually inhibitory feedback loop between two self-activating master regulators A and B, also called as toggle switch. Typically, it can allow for three stable states - (high A, low B), (low A, high B) and (medium A, medium B). A toggle triad - three mutually repressing regulators A, B and C, i.e. three toggle switches arranged circularly (between A and B, between B and C, and between A and C) - can allow for six stable states: three single positive and three double positive ones. However, the operating principles of larger toggle polygons, i.e. toggle switches arranged circularly to form a polygon, remain unclear. Here, we simulate using both discrete and continuous methods the dynamics of different sized toggle polygons. We observed a pattern in their steady state frequency depending on whether the polygon was an even or odd numbered one. The even-numbered toggle polygons result in two dominant states with consecutive components of the network expressing alternating high and low levels. The odd-numbered toggle polygons, on the other hand, enable more number of states, usually twice the number of components with the states that follow circular permutation patterns in their composition. Incorporating self-activations preserved these trends while increasing the frequency of multistability in the corresponding network. Our results offer insights into design principles of circular arrangement of regulatory units involved in cell-fate decision making, and can offer design strategies for synthesizing genetic circuits.

systems biology↗

bioRxiv2020

Scalable production of tissue-like vascularised liver organoids from human PSCs.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single-cell RNA sequencing and immunofluorescence we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage - the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a pre-requisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.

cell biology↗

bioRxiv2021

Postembryonic development of the two-spotted field cricket (Gryllus bimaculatus): a staging system

The two-spotted field cricket Gryllus bimaculatus has emerged as a central model for studies on insect development, regeneration, and physiology. G. bimaculatus has the most sophisticated functional genetic toolkit of any hemimetabolous insect, making it a foremost model to understand the evolutionary developmental biology and comparative physiology of insects. However, the morphology and stages of postembryonic development have never been comprehensively reported. Here, 8 morphologically defined stages are described. Size, coloration, and the morphology of wing buds, hind tibial spines, and the ovipositor are the best landmarks for staging. The stages correspond to the 8-12 moult-based instars present in the literature. The staging system aims to standardise studies on the postembryonic development of G. bimaculatus and serve as a point of reference for delineating interspecific postembryonic homologies within Orthoptera.

zoology↗

bioRxiv2021

CBMOS: a GPU-enabled Python framework for the numerical study of center-based models

Cell-based models are becoming increasingly popular for applications in developmental biology. However, the impact of numerical choices on the accuracy and efficiency of the simulation of these models is rarely meticulously tested. We present CBMOS, a Python framework for the simulation of the center-based or cell-centered model. Contrary to other implementations, CBMOS focus is on facilitating numerical study of center-based models by providing access to multiple ODE solvers and force functions through a flexible, user-friendly API. We show-case its potential by evaluating the use of the backward Euler method for calculating the trajectories of two-dimensional cell populations. We confirm that although for moderate accuracy levels the backward Euler method allows for larger time step sizes than the commonly used forward Euler method, its additional computational cost due to being an implicit method prohibits its use for practical test cases. CBMOS is available on GitHub1 and PyPI under an MIT license. It allows for fast prototyping on a CPU for small systems through the use of NumPy. Using CuPy on a GPU, cell populations of up to 10,000 cells can be simulated within a few seconds. As such, we hope it can also be of use to modelers interested in testing preliminary hypotheses before committing to more complex center-based model frameworks. AMS subject classification65Z05, 92C15, 92-10

biophysics↗

bioRxiv2021

A contraction-reaction-diffusion model for circular pattern formation in embryogenesis

Reaction-diffusion models have been widely used to elucidate pattern formation in developmental biology. More recently, they have also been applied in modeling cell fate patterning that mimic early-stage human development events utilizing geometrically confined pluripotent stem cells. However, the traditional reaction-diffusion equations could not satisfactorily explain the concentric ring distributions of various cell types, as they do not yield circular patterns even for circular domains. In previous mathematical models that yield ring patterns, certain conditions that lack biophysical understandings had been considered in the reaction-diffusion models. Here we hypothesize that the circular patterns are the results of the coupling of the mechanobiological factors with the traditional reaction-diffusion model. We propose two types of coupling scenarios: tissue tension-dependent diffusion flux and traction stress-dependent activation of signaling molecules. By coupling reaction-diffusion equations with the elasticity equations, we demonstrate computationally that the contraction-reaction-diffusion model can naturally yield the circular patterns.

bioengineering↗

bioRxiv2021

Reactive/Non-cooperative individuals advance Population's Synchronization: Modeling of Dictyostelium discoideum Concerted Signaling during Aggregation Phase

Orchestrated chemical signaling of single cells sounds to be a linchpin of emerging organization and multicellular life form. The social amoeba Dictiostelium discoiudium is a well-studied model organism to explore overall pictures of grouped behavior in developmental biology. The chemical waves secreted by aggregating Dictiostelium is a superb example of pattern formation. The waves are either circular or spiral in shape, according to the incremental population density of a self-aggregating community of individuals. Here, we revisit the spatiotemporal patterns that appear in an excitable medium due to synchronization of randomly firing individuals, but with a more parsimonies attitude. According to our model, a fraction of these individuals is refusal to amplify external stimulants. Our simulations indicate that the cells enhance the systems asymmetry and as a result, nucleate early sustainable spiral territory zones, provided that their relative population does not exceed a tolerable threshold.

biophysics↗

bioRxiv2021

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data

Identity and functions of plant cells are influenced by their precise cellular location within the plant body. Cellular heterogeneity in growth and differentiation trajectories results in organ patterning. Therefore, assessing this heterogeneity at molecular scale is a major question in developmental biology. Single-cell transcriptomics (scRNA-seq) allows to characterize and quantify gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during the scRNA-seq procedure. To recover the original location of cells is essential to link gene activity with cellular function and morphology. Here, we reconstruct genome-wide gene expression patterns of individual cells in a floral meristem by combining single-nuclei RNA-seq with 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the data are used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com

plant biology↗

bioRxiv2021

Functional constraints on insect immune system components govern their evolutionary trajectories

The role of constraints in shaping evolutionary outcomes is often investigated in the contexts of developmental biology and population genetics, in terms of the capacity to generate new variants as well as how selection either limits or promotes consequent phenotypic change. Comparative genomics also recognises the role of constraints, in terms of shaping the evolution of gene and genome architectures, sequence evolutionary rates, and gene gains and losses, as well as on molecular phenotypes. Characterising patterns of genomic change where putative functions and interactions of system components are relatively well-described offers opportunities to explore whether genes with similar or analogous roles exhibit similar evolutionary trajectories, possibly governed by common constraints. Using insect innate immunity as our study system, we hypothesise that quantitative characterisation of gene evolutionary histories can define distinct dynamics associated with different functional roles. We develop metrics that quantify gene evolutionary histories, employ these to characterise evolutionary features of immune gene repertoires, and explore relationships between gene family evolutionary profiles and their roles in immunity to understand how different constraints may relate to distinct dynamics. We identified three main axes of evolutionary trajectories characterised by gene duplication and synteny, maintenance/stability and sequence conservation, and loss and sequence divergence, highlighting similar and contrasting patterns across these axes amongst subsets of immune genes. Our results indicate that where and how genes participate in immune responses limit the range of possible evolutionary scenarios they exhibit. Comparative genomics approaches therefore offer opportunities to characterise how functional constraints on different components of biological systems govern their evolutionary trajectories.

evolutionary biology↗

bioRxiv2021

Topological properties accurately predict cell division events and organization of Arabidopsis thaliana's shoot apical meristem

Cell division and the resulting changes to the cell organization affect the shape and functionality of all tissues. Thus, understanding the determinants of the tissue-wide changes imposed by cell division is a key question in developmental biology. Here, we use a network representation of live cell imaging data from shoot apical meristems (SAMs) in Arabidopsis thaliana to predict cell division events and their consequences at a tissue level. We show that a classifier based on the SAM network properties is predictive of cell division events, with validation accuracy of 82%, on par with that based on cell size alone. Further, we demonstrate that the combination of topological and biological properties, including: cell size, perimeter, distance, and shared cell wall between cells, can further boost the prediction accuracy of resulting changes in topology triggered by cell division. Using our classifiers, we demonstrate the importance of microtubule mediated cell-to-cell growth coordination in influencing tissue-level topology. Altogether, the results from our network-based analysis demonstrates a feedback mechanism between tissue topology and cell division in A. thalianas SAMs. Summary statementwe use a network representation of live cell imaging data from SAMs in Arabidopsis thaliana to predict cell division events and their consequences at a tissue level.

systems biology↗

bioRxiv2021

Genome of the bi-annual Rio Pearlfish (Nematolebias whitei), a killifish model species for Eco-Evo-Devo

The Rio Pearlfish Nematolebias whitei is a bi-annual killifish species inhabiting seasonal pools of the Rio de Janeiro region that dry twice per year. Embryos enter dormant diapause stages in the soil, waiting for the inundation of the habitat which triggers hatching and commencement of a new life cycle. This species represents a convergent, independent origin of annualism from other emerging killifish model species. While some transcriptomic datasets are available for Rio Pearlfish, thus far a sequenced genome has been unavailable. Here we present a high quality, 1.2Gb chromosome-level genome assembly, genome annotations and a comparative genomic investigation of the Rio Pearlfish as representative of a vertebrate clade that evolved environmentally-cued hatching. We show conservation of 3-D genome structure across teleost fish evolution, developmental stages, tissues and cell types. Our analysis of mobile DNA shows that Rio Pearlfish, like other annual killifishes, possesses an expanded transposable element profile with implications for rapid aging and adaptation to harsh conditions. We use the Rio Pearlfish genome to identify its hatching enzyme gene repertoire and the location of the hatching gland, a key first step in understanding the developmental genetic control of hatching. The Rio Pearlfish genome expands the comparative genomic toolkit available to study convergent origins of seasonal life histories, diapause, and rapid aging phenotypes. We present the first set of genomic resources for this emerging model organism, critical for future functional genetic and multi-omic explorations of "Eco-Evo-Devo" phenotypes in resilience and adaptation to extreme environments. Significance: Seasonal or annual killifishes are emerging models for aging, life history adaptions to extreme environments, and ecological evolutionary developmental biology (Eco-Evo-Devo). Most studies have thus far focused on the African turquoise killifish Nothobranchius furzeri and the South American Austrofundulus limneaus. We sequenced and analyzed the genome of the Rio Pearlfish Nematolebias whitei from the Rio de Janeiro region, a seasonal species representing a convergent origin of seasonality from other sequenced killifish species, strengthening the comparative potential of Aplocheiloid killifishes as a model clade for the comparative and functional genomics of animal resilience to environmental change.

genomics↗

bioRxiv2021

Variation of female pronucleus reveal oocyte or embryo abnormality: An expert experience deep learning of non-dark box analysis

BackgroundPronuclear assessment appears to have the ability to distinguish good and bad embryos in the zygote stage, but paradoxical results were obtained in clinical studies. This situation might be caused by the robust qualitative detection of the development of dynamic pronuclei. Here, we aim to establish a quantitative pronuclear measurement method by applying expert experience deep learning from large annotated datasets. MethodsConvinced handle-annotated 2PN images (13419) were used for deep learning then corresponded errors were recorded through handle check for subsequent parameters adjusting. We used 790 embryos with 52479 PN images from 155 patients for analysis the area of pronuclei and the pre-implantation genetic test results. Establishment of the exponential fitting equation and the key coefficient {beta} 1was extracted from the model for quantitative analysis for pronuclear(PN) annotation and automatic recognition. FindingsBased on the female original PN coefficient {beta}1, the chromosome-normal rate in the blastocyst with biggest PN area is much higher than that of the blastocyst with smallest PN area (58.06% vs. 45.16%, OR=1.68 [1.07-2.64]; P=0.031). After adjusting coefficient {beta}1 by the first three frames which high variance of outlier PN areas was removed, coefficient {beta}1 at 12 hours and at 14 hours post-insemination, similar but stronger evidence was obtained. All these discrepancies resulted from the female propositus in the PGT-SR subgroup and smaller chromosomal errors. Conclusion(s)The results suggest that detailed analysis of the images of embryos could improve our understanding of developmental biology. FundingNone

genetics↗

bioRxiv2021

Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

cell biology↗