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Results for “developmental biology”

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bioRxiv2016

Resurrected protein interaction networks reveal the strong rewiring that leads to network organisation after whole genome duplication

The evolution of plants is characterized by several rounds of ancient whole genome duplication, sometimes closely associated with the origin of large groups of species. A good example is the {gamma} triplication at the origin of core eudicots. Core eudicots comprise about 75% of flowering plants and are characterized by the canalization of reproductive development. To better understand the impact of this genomic event, we studied the protein interaction network of MADS-domain transcription factors, which are key regulators of reproductive development. We accurately inferred, resurrected and tested the interactions of ancestral proteins before and after the triplication and directly compared these ancestral networks to the networks of Arabidopsis and tomato. We find that the {gamma} triplication generated a dramatically innovated network that strongly rewired through the addition of many new interactions. Many of these interactions were established between paralogous proteins and a new interaction partner, establishing new redundancy. Simulations show that both node and edge addition through the triplication were important to maintain modularity in the network. In addition to generating insights into the impact of whole genome duplication and elementary processes involved in network evolution, our data provide a resource for comparative developmental biology in flowering plants.

Evolutionary Biology↗

bioRxiv2017

The origin of animal multicellularity and cell differentiation

How animals evolved from their single-celled ancestors over 600 million years ago is poorly understood. Comparisons of genomes from animals and their closest relatives - choanoflagellates, filastereans and ichthyosporeans - have recently revealed the genomic landscape of animal origins. However, the cell and developmental biology of the first animals have been less well examined. Using principles from evolutionary cell biology, we reason that the last common ancestor of animals and choanoflagellates (the Urchoanozoan) used a collar complex - a flagellum surrounded by a microvillar collar - to capture bacterial prey. The origin of animal multicellularity likely occurred through the modification of pre-existing mechanisms for extracellular matrix synthesis and regulation of cytokinesis. The progenitors of animals likely developed clonally through serial division of flagellated cells, giving rise to sheets of cells that folded into spheres by a morphogenetic process comparable to that seen in modern choanoflagellate rosettes and calcareous sponge embryos. Finally, we infer that cell differentiation evolved in the animal stem-lineage by a combination of three mechanisms: division of labor from ancient plurifunctional cell types, conversion of temporally segregated phenotypes into spatially segregated cell types, and functional innovation.

evolutionary biology↗

bioRxiv2017

Integrated transcriptome and epigenome analyses identify alternative splicing as a novel candidate linking histone modifications to embryonic stem cell fate decision

BackgroundUnderstanding the embryonic stem cell (ESC) fate decision between self-renewal and proper differentiation is important for developmental biology and regenerative medicine. Attention has focused on mechanisms involving histone modifications, alternative pre-mRNA splicing, and cell-cycle progression. However, their intricate interrelations and joint contributions to ESC fate decision remain unclear.\n\nResultsWe analyze the transcriptomes and epigenomes of human ESC and five types of differentiated cells. We identify thousands of alternatively spliced exons and reveal their development and lineage-dependent characterizations. Several histone modifications show dynamic changes in alternatively spliced exons and three are strongly associated with 52.8% of alternative splicing events upon hESC differentiation. The histone modification-associated alternatively spliced genes predominantly function in G2/M phases and ATM/ATR-mediated DNA damage response pathway for cell differentiation, whereas other alternatively spliced genes are enriched in the G1 phase and pathways for self-renewal. These results imply a potential epigenetic mechanism by which some histone modifications contribute to ESC fate decision through the regulation of alternative splicing in specific pathways and cell-cycle genes. Supported by experimental validations and extended dataset from Roadmap/ENCODE projects, we exemplify this mechanism by a cell cycle-related transcription factor, PBX1, which regulates the pluripotency regulatory network by binding to NANOG. We suggest that the isoform switch from PBX1a to PBX1b links H3K36me3 to hESC fate determination through the PSIP1/SRSF1 adaptor, which results in the exon skipping of PBX1.\n\nConclusionWe reveal the mechanism by which alternative splicing links histone modifications to stem cell fate decision.

bioinformatics↗

bioRxiv2017

Independent erosion of conserved transcription factor binding sites points to shared hindlimb, vision, and scrotum loss in different mammals

Genetic variation in cis-regulatory elements is thought to be a major driving force in morphological and physiological change. However, identifying transcription factor binding events which code for complex traits remains a challenge, motivating novel means of detecting putatively important binding events. Using a curated set of 1,154 high-quality transcription factor motifs, we demonstrate that independently eroded binding sites are enriched for independently lost traits in three distinct pairs of placental mammals. We show that these independently eroded events pinpoint the loss of hindlimbs in dolphin and manatee, degradation of vision in naked mole-rat and star-nosed mole, and the loss of scrotum in white rhinoceros and Weddell seal. Our study exhibits a novel methodology to detect cis-regulatory mutations which help explain a portion of the molecular mechanism underlying complex trait formation and loss.\n\nAuthor SummaryEvolution has produced an astounding variety of species with incredibly diverse phenotypes. A central question in evolutionary developmental biology is how (and which) DNA evolves to encode all of these different traits. A prevailing hypothesis is that changes in regulatory DNA, short stretches of DNA which control the expression of protein-coding genes, drive important differences in trait formation between species. The basic building block of regulatory DNA is thought to be transcription factor binding sites, shortl genomic sequences which attract proteins whose central role is to control the rate of transcription. In this study, we asked whether the independent erosion of otherwise highly conserved transcription factor binding sites points to a trait shared between species which have undergone similar adaptations. We show that our method is able to point to the loss of hindlimbs in dolphin and manatee, poor vision in naked mole-rat and star-nosed mole, and loss of scrotum in Weddell seal and white rhinoceros. Overall, our study exhibits a means of detecting evolutionarily important genomic regions which help explain a portion of complex trait loss and retention.

evolutionary biology↗

bioRxiv2020

Computational modelling of cambium activity provides a regulatory framework for simulating radial plant growth

Precise organization of growing structures is a fundamental process in developmental biology. In plants, radial growth is mediated by the cambium, a stem cell niche continuously producing wood (xylem) and bast (phloem) in a strictly bidirectional manner. While this process contributes large parts to terrestrial biomass, cambium dynamics eludes direct experimental access due to obstacles in live cell imaging. Here, we present a cell-based computational model visualizing cambium activity and integrating the function of central cambium regulators. Performing iterative comparisons of plant and model anatomies, we conclude that the receptor- like kinase PXY and its ligand CLE41 are part of a minimal framework sufficient for instructing tissue organization. By integrating tissue-specific cell wall stability values, we moreover probe the influence of physical constraints on tissue geometry. Our model highlights the role of intercellular communication within the cambium and shows that a limited number of factors is sufficient to create radial growth by bidirectional tissue production. Impact statementRadial plant growth produces large parts of terrestrial biomass and can be computationally simulated with the help of an instructive framework of intercellular communication loops.

plant biology↗

bioRxiv2020

Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix

Epithelial sheets play important roles in defining organ architecture during development. Here, we employed an iterative experimental and multi-scale computational modeling approach to decouple direct and indirect effects of actomyosin-generated forces, nuclear positioning, extracellular matrix (ECM), and cell-cell adhesion in shaping Drosophila wing imaginal discs, a powerful system for elucidating general principles of epithelial morphogenesis. Basally generated actomyosin forces are found to regulate apically biased nuclear positioning and are required for generating epithelial bending and cell elongation of the wing disc pouch. Surprisingly, however, short-term pharmacological inhibition of ROCK-driven actomyosin contractility does not impact the maintenance of tissue height or curved shape. In comparison, the relative tautness of the extracellular basement membrane is also patterned between regions of the wing disc. However, computational simulations show that patterning of ECM tautness provides only a minor contribution to modulating tissue shape. Instead, the buildup of a passive ECM pre-strain serves a principle role in shape maintenance. Surprisingly, this is independent from the maintenance of actomyosin contractility. Furthermore, localized apical adhesion between the two cell layers within the wing disc requires ROCK-driven actomyosin activity in the absence of the basal extracellular matrix. This apical adhesion between the two cell layers provides additional mechanical support to help maintain tissue integrity. The combined experimental and computational approach provides general insight into how the subcellular forces are generated and maintained within individual cells to induce tissue curvature and suggests an important design principle of epithelial organogenesis whereby forces generated by actomyosin followed by maintenance as pre-strain within the ECM are interconnected, but functionally separable. Significance statementA major outstanding question in developmental biology is the elucidation of general principles of organ shape formation and maintenance. Here, an iterative experimental and multi-scale computational modeling approach reveals that actomyosin contractility generates the bent profile along the anterior-posterior axis while tension within the ECM is sufficient and necessary for preserving the bent shape even in the absence of continued actomyosin contractility once the shape is generated. The mechanisms tested in this study define the necessary factors for establishing the shape of the wing disc, which later everts to form the adult wing during pupal development. The method can be extended to test novel mechanisms of other epithelial systems that consist of several cellular and ECM layers.

biophysics↗

bioRxiv2020

TiFoSi: an Efficient Tool for Mechanobiology Simulations of Epithelia

AboutThis document is an extended version of the main text where some details and results are fleshed out. Further details can be also found in the manual of the code and at TiFoSis website: http://tifosi.thesimbiosys.com. MotivationEmerging phenomena in developmental biology and tissue engineering are the result of feedbacks between gene expression and cell biomechanics. In that context, in silico experiments are a powerful tool to understand fundamental mechanisms and to formulate and test hypotheses. ResultsHere we present TiFoSi, a computational tool to simulate the cellular dynamics of planar epithelia. TiFoSi allows to model feedbacks between cellular mechanics and gene expression (either in a deterministic or a stochastic way), the interaction between different cell populations, the custom design of the cell cycle and cleavage properties, the protein number partitioning upon cell division, and the modeling of cell communication (juxtacrine and paracrine signalling). TiFoSi fills a niche in the field of software solutions to simulate the mechanobiology of epithelia because of its functionalities, computational efficiency, and its user-friendly approach to design in silico experiments using XML configuration files. Availabilityhttp://tifosi.thesimbiosys.com Contactjbuceta@lehigh.edu

systems biology↗

bioRxiv2020

Clinically oriented system-based embryology; a significant course in clinical practice

IntroductionEmbryology is a branch of medical sciences in developmental biology. Since the knowledge of embryology is of special significance for medical students, this study was conducted with the purpose of elucidating the viewpoint of medical students of Zanjan University of Medical Sciences in Iran on the application of embryology courses in fulfilling clinical purposes. MethodsThis cross-sectional study was conducted in 2018-2019 with a census method on all clinical medical students (trainees and interns). To collect medical students opinions, the researcher-designed questionnaire was used. The validity of the questionnaire was confirmed by embryology experts by content validity ratio (CVR) and factor analysis and the reliability of the questionnaire Cronbachs alpha coefficient (0.9), respectively. Data were analyzed by SPSS (version 24) and Lisrel software. Values with P<0.05 were considered significant. ResultsDescriptive statistics in the field of general and system-based embryology demonstrated that the topics of birth defects and development of body cavities (diaphragm development) have the most influence on clinical practice of medical students. ConclusionsIt seems that more focus on system-based embryology courses leads to a better performance of medical students in clinical courses.

scientific communication and education↗

bioRxiv2020

Widefield light sheet microscopy using an Airy beam combined with deep-learning super-resolution

Imaging across length scales and in depth has been an important pursuit of widefield optical imaging. This promises to reveal fine cellular detail within a widefield snapshot of a tissue sample. Current advances often sacrifice resolution through selective sub-sampling to provide a wide field of view in a reasonable time scale. We demonstrate a new avenue for recovering high-resolution images from sub-sampled data in light-sheet microscopy using deep-learning super-resolution. We combine this with the use of a widefield Airy beam to achieve high-resolution imaging over extended fields of view and depths. We characterise our method on fluorescent beads as test targets. We then demonstrate improvements in imaging amyloid plaques in a cleared brain from a mouse model of Alzheimers disease, and in excised healthy and cancerous colon and breast tissues. This development can be widely applied in all forms of light sheet microscopy to provide a two-fold increase in the dynamic range of the imaged length scale. It has the potential to provide further insight into neuroscience, developmental biology and histopathology.

biophysics↗

bioRxiv2020

Integration of anatomy ontology data with protein-protein interaction networks improves the candidate gene prediction accuracy for anatomical entities

BackgroundIdentification of genes responsible for anatomical entities is a major requirement in many fields including developmental biology, medicine, and agriculture. Current wet-lab techniques used for this purpose, such as gene knockout, are high in resource and time consumption. Protein-protein interaction (PPI) networks are frequently used to predict disease genes for humans and gene candidates for molecular functions, but they are rarely used to predict genes for anatomical entities. This is because PPI networks suffer from network quality issues, which can be a limitation for their usage in predicting candidate genes for anatomical entities. We developed an integrative framework to predict candidate genes for anatomical entities by combining existing experimental knowledge about gene-anatomy relationships with PPI networks using anatomy ontology annotations. We expected this integration to improve the quality of the PPI networks and be better optimized to predict candidate genes for anatomical entities. We used existing Uberon anatomy entity annotations for zebrafish and mouse genes to construct gene networks by calculating semantic similarity between the genes. These anatomy-based gene networks are semantic networks, as they are constructed based on the Uberon anatomy ontology annotations that are obtained from the experimental data in the literature. We integrated these anatomy-based gene networks with mouse and zebrafish PPI networks retrieved from the STRING database, and we compared the performance of their network-based candidate gene predictions. ResultsAccording to candidate gene prediction performance evaluations tested under four different semantic similarity calculation methods (Lin, Resnik, Schlicker, and Wang), the integrated networks showed better receiver operating characteristic (ROC) and precision-recall curve performances than PPI networks for both zebrafish and mouse. ConclusionIntegration of existing experimental knowledge about gene-anatomical entity relationships with PPI networks via anatomy ontology improves the network quality, which makes them better optimized for predicting candidate genes for anatomical entities.

bioinformatics↗

bioRxiv2020

Impact of force function formulations on the numerical simulation of centre-based models

Centre-based, or cell-centre models are a framework for the computational study of multicellular systems with widespread use in cancer modelling and computational developmental biology. At the core of these models are the numerical method used to update cell positions and the force functions that encode the pairwise mechanical interactions of cells. For the latter there are multiple choices that could potentially affect both the biological behaviour captured, and the robustness and efficiency of simulation. For example, available open-source software implementations of centre-based models rely on different force functions for their default behaviour and it is not straightforward for a modeler to know if these are interchangeable. Our study addresses this problem and contributes to the understanding of the potential and limitations of three popular force functions from a numerical perspective. We show empirically that choosing the force parameters such that the relaxation time for two cells after cell division is consistent between different force functions results in good agreement of the population radius of a growing monolayer. Furthermore, we report that numerical stability is not sufficient to prevent unphysical cell trajectories following cell division, and consequently, that too large time steps can cause geometrical differences at the population level.

biophysics↗

bioRxiv2020

Clearing-induced tissue shrinkage: Novel observation of a thickness size effect.

The use of clearing agents has provided new insights in various fields of medical research (developmental biology, neurology) by enabling examination of tissue architecture in 3D. One of the challenges is that clearing agents induce tissue shrinkage and the shrinkage rates reported in the literature are incoherent. Here, we report that the shrinkage for a widely-used clearing agent decreases significantly with increasing sample size, and report an analytical description.

biophysics↗

bioRxiv2020

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

Levels of the second messenger bis-3-5-cyclic di-guanosinemonophosphate (c-di-GMP) determine when Streptomyces initiate sporulation to survive under adverse conditions. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor {sigma}WhiG. However, functions of the development-specific c-di-GMP diguanylate cyclases (DGCs) CdgB and CdgC, and the phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Global transcriptome analysis of {Delta}cdgB, {Delta}cdgC, {Delta}rmdA and {Delta}rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20 % of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores. Altogether, this study provides a global view of the c-di-GMP-dependent genes that contribute to the hyphae-to-spores transition and sheds light on the shared and specific functions of the key enzymes involved in c-di-GMP metabolism in S. venezuelae. ImportanceStreptomyces are important producers of clinical antibiotics. The ability to synthesize these natural products is connected to their developmental biology, which includes a transition from filamentous cells to spores. The widespread bacterial second messenger c-di-GMP controls this complex switch and is a promising tool to improve antibiotic production. Here, we analyzed the enzymes that make and break c-di-GMP in S. venezuelae by studying the genome-wide transcriptional effects of the DGCs CdgB and CdgC and the PDEs RmdA and RmdB. We found that the c-di-GMP specified by these enzymes has a global regulatory role. However, despite shared enzymatic activities, the four c-di-GMP enzymes have specialized inputs into differentiation. Altogether, we demonstrate that altering c-di-GMP levels through the action of selected enzymes yields characteristically distinct transcriptional profiles; this can be an important consideration when modulating c-di-GMP for the purposes of natural product synthesis in Streptomyces.

microbiology↗

bioRxiv2020

Spatiotemporal single-cell RNA sequencing of developing hearts reveals interplay between cellular differentiation and morphogenesis

Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about cellular interactions and tissue morphology. Here, we combined single-cell and spatial transcriptomics with new algorithms for data integration to study the early development of the chicken heart. We collected data from four key ventricular development stages, ranging from the early chamber formation stage to the late four-chambered stage. We created an atlas of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions revealed the intricate interplay between cellular differentiation and morphogenesis in cardiac cellular lineages. Using spatially resolved expression analysis, we identified anatomically restricted gene expression programs. Last, we discovered a stage-dependent role for the small secreted peptide, thymosin beta-4, in the coordination of multi-lineage cellular populations. Overall, our study identifies key stage-specific regulatory programs that govern cardiac development.

genomics↗

bioRxiv2020

Insights on the evolution of vertebrate microbiomes from the analysis of the Xenopus frog microbiota across life stages

BackgroundThe microorganism world living in amphibians is still largely under-represented and under-studied in the literature. Among anuran amphibians, African clawed frogs of the Xenopus genus stand as well-characterized models with an in-depth knowledge of their developmental biological processes including their metamorphosis. In this study, we analyzed the succession of microbial communities and their activities across diverse body habitats of Xenopus tropicalis using different approaches including flow cytometry and 16s rDNA gene metabarcoding. We also evaluated the metabolic capacity of the premetamorphic tadpoles gut microbiome using metagenomic and metatranscriptomic sequencing. ResultsWe analyzed the bacterial components of the Xenopus gut microbiota, the adult gut biogeography, the succession of communities during ontogeny, the impact of the alimentation in shaping the tadpoles gut bacterial communities and the transmission of skin and fecal bacteria to the eggs. We also identified the most active gut bacteria and their metabolic contribution to tadpole physiology including carbohydrate breakdown, nitrogen recycling, essential amino-acids and vitamin biosynthesis. ConclusionsWe present a comprehensive new microbiome dataset of a laboratory amphibian model. Our data provide evidences that studies on the Xenopus tadpole model can shed light on the interactions between a vertebrate host and its microbiome. We interpret our findings in light of bile acids being key molecular components regulating the gut microbiome composition during amphibian development and metamorphosis. Further studies into the metabolic interactions between amphibian tadpoles and their microbiota during early development and metamorphosis should provide useful information on the evolution of host-microbiota interactions in vertebrates. This article has been peer-reviewed and recommended by Peer Community in Genomics https://doi.org/10.24072/pci.genomics.100005

genomics↗

bioRxiv2020

3D bioprinting of co-cultured osteogenic spheroids for bone tissue fabrication

Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds have been widely used in bone engineering applications. However, scaffold-based bone tissue constructs have had limited clinical translation due to constrains in supporting scaffolds, minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up method, inspired from embryonic developmental biology, to build three-dimensional (3D) scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) (hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 days, with enhanced osteogenic differentiation and cell viability in the core of the spheroids compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new bioprinting technique which allows precise positioning of spheroids (11% with respect to the spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible changes in their shape for two days after bioprinting owing to the reduced proliferative potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with actin-filament formation and high expression of osteogenic and endothelial-specific gene factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling.

bioengineering↗

bioRxiv2020

Post-mitotic myotubes repurpose the cytokinesis machinery to effect cellular guidance

Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. Here we show the mitotic kinase Polo, which regulates all steps of mitosis [1-3], also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis [4-6]. We show that in the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases.

cell biology↗

bioRxiv2020

MAGIC: Mosaic Analysis by gRNA-Induced Crossing-over

Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for one chromosome arm, we demonstrate the method’s application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites in the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can be applied in any organism that is compatible with CRISPR/Cas9.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗