Visualization Of Gene Expression Research Articles

The human infertility single-cell testis atlas (HISTA): an interactive molecular scRNA-Seq reference of the human testis.

Single-cell RNA-seq (scRNA-Seq) has been widely adopted to study gene expression of the human testis. Several datasets of scRNA-Seq from human testis have been generated from different groups processed with different informatics pipelines. An integrated atlas of scRNA-Seq expression constructed from multiple donors, developmental ages, and fertility states would be widely useful for the testis research community. To describe the generation and use of the human infertility single-cell testis atlas (HISTA), an interactive web tool for understanding human spermatogenesis through scRNA-Seq analysis. We obtained scRNA-Seq datasets derived from 12 donors, including healthy adult controls, juveniles, and several infertility cases, and reprocessed these data using methods to remove batch effects. Using Shiny, an open-source environment for data visualization, we created numerous interactive tools for exploring the data, some of which support simple statistical hypothesis testing. We used the resulting HISTA browser and its underlying data to demonstrate HISTA's value for testis researchers. A primary application of HISTA is to search by a single gene or a set of genes; thus, we present various analyses that quantify and visualize gene expression across the testis cells and pathology. HISTA also contains machine-learning-derived gene modules ("components") that capture the entire transcriptional landscape of the testis tissue. We show how the use of these components can simplify the highly complex data in HISTA and assist with the interpretation of genes with unknown functions. Finally, we demonstrate the diverse ways HISTA can be used for new data analysis, including hypothesis testing. HISTA is a research environment that can help scientists organize and understand the high-dimensional transcriptional landscape of the human testis. HISTA has already contributed to published testis research and can be updated as needed with input from the research community or downloaded and modified for individual needs.

HBPGEO: AN APPLICATION SYSTEM FOR ANALYZING AND VISUALIZING GENE EXPRESSION DATA OF HYPERTENSION

The analysis of gene expression data using existing methods and tools has become a fundamental approach for researchers to explore the mechanisms of hypertension. However, many researchers lack programming skills or statistical knowledge. Although gene expression data is widely available, they originate from various experiments with different standards and quality levels, causing inconvenience to researchers. While tools and databases with interactive interfaces exist for data analysis, they may have limited applicability. This study aimed to construct High Blood Pressure Gene Expression Omnibus (HBPGEO), a program based on the R Shiny platform. It integrated the discovery of differentially expressed genes, hub gene identification, gene function analysis, and more. It allowed users to select suitable methods and set different parameters during analysis, visualize results interactively, and download the analyzed results. The study successfully utilized HBPGEO to identify hypertension-related biomarkers.

Design of a targeted blood transcriptional panel for monitoring immunological changes accompanying pregnancy.

Immunomodulatory processes exert steering functions throughout pregnancy. Detecting diversions from this physiologic immune clock may help identify pregnant women at risk for pregnancy-associated complications. We present results from a data-driven selection process to develop a targeted panel of mRNAs that may prove effective in detecting pregnancies diverting from the norm. Based on a de novo dataset from a resource-constrained setting and a dataset from a resource-rich area readily available in the public domain, whole blood gene expression profiles of uneventful pregnancies were captured at multiple time points during pregnancy. BloodGen3, a fixed blood transcriptional module repertoire, was employed to analyze and visualize gene expression patterns in the two datasets. Differentially expressed genes were identified by comparing their abundance to non-pregnant postpartum controls. The selection process for a targeted gene panel considered (i) transcript abundance in whole blood; (ii) degree of correlation with the BloodGen3 module; and (iii) pregnancy biology. We identified 176 transcripts that were complemented with eight housekeeping genes. Changes in transcript abundance were seen in the early stages of pregnancy and similar patterns were observed in both datasets. Functional gene annotation suggested significant changes in the lymphoid, prostaglandin and inflammation-associated compartments, when compared to the postpartum controls. The gene panel presented here holds promise for the development of predictive, targeted, transcriptional profiling assays. Such assays might become useful for monitoring of pregnant women, specifically to detect potential adverse events early. Prospective validation of this targeted assay, in-depth investigation of functional annotations of differentially expressed genes, and assessment of common pregnancy-associated complications with the aim to identify these early in pregnancy to improve pregnancy outcomes are the next steps.

An eFP reference gene expression atlas for mangosteen

Mangosteen is the queen of fruits with many health benefits and high pharmaceutical potential but remains an underutilized crop. There are several reports of mangosteen organelle genomes and transcriptomes from various tissues but a reference genome or transcriptome is still lacking. In this study, we aimed to generate a publicly accessible reference transcriptome of Garcinia mangostana variety Manggis (local fruit in Southeast Asia) to facilitate research in mangosteen functional genomics. De novo assembly was performed using Trinity with all the Illumina sequence datasets and generated 369,861 transcripts with an N50 of 1,433 bp. SuperTranscripts and TGICL transcript clustering approaches were taken to reduce redundant transcripts, in which the latter generated a more comprehensive reference transcriptome with a higher N50 value, Benchmarking Universal Single-Copy Orthologs (BUSCO) score, and read mapping rates. A total of 118,165 (43.7 %) unigenes were functionally annotated. To visualize gene expression across different mangosteen tissues, developmental stages, and experiments based on the reference transcriptome, we constructed a mangosteen electronic Fluorescent Pictograph (eFP) browser. This allows users to easily visualize the expression of genes in absolute, relative, and compare modes. In addition, researchers can perform online BLAST search against the in-house BAR SequenceServer for homologous sequences that match mangosteen transcripts to explore corresponding expression patterns via direct links to the eFP browser. This reference transcriptome and eFP browser (accessible at https://bar.utoronto.ca/efp_mangosteen/cgi-bin/efpWeb.cgi) provide a useful online tool for future research and improvement of mangosteen.

Developmental and environmental plasticity in opsin gene expression in Lake Victoria cichlid fish.

In many organisms, sensory abilities develop and evolve according to the changing demands of navigating, foraging, and communication across different environments and life stages. Teleost fish inhabit heterogeneous light environments and exhibit a large diversity in visual system properties among species. Cichlids are a classic example of this diversity; visual system variation is generated by different tuning mechanisms that involve both genetic factors and phenotypic plasticity. Here, we document the developmental progression of visual pigment gene expression in Lake Victoria cichlids and test if these patterns are influenced by variation in light conditions. We reared two sister species of Pundamilia to adulthood in two distinct visual conditions that resemble the light environments that they naturally inhabit in Lake Victoria. We also included interspecific first-generation hybrids. We focused on the four opsins that are expressed in Pundamilia adults (using real-time quantitative polymerase chain reaction (RT-qPCR)) (SWS2B, SWS2A, RH2A, and LWS) at 17 time points. We find that opsin expression profiles progress from shorter-wavelength sensitive opsins to longer-wavelength sensitive opsins with increasing age, in both species and their hybrids. The developmental trajectories of opsin expression also responded plastically to the visual conditions. Developmental and environmental plasticity in opsin expression may provide an important stepping stone in the evolution of cichlid visual system diversity.

Clearing of Vascular Tissue in Arabidopsis thaliana for Reporter Analysis of Gene Expression.

To study the gene regulatory mechanisms modulating development is essential to visualize gene expression patterns at cellular resolution. However, this kind of analysis has been limited as a consequence of the plant tissues' opacity. In the last years, ClearSee has been increasingly used to obtain high-quality imaging of plant tissue anatomy combined with the visualization of gene expression patterns. ClearSee is established as a major tissue clearing technique due to its simplicity and versatility.In this chapter, we outline an easy-to-follow ClearSee protocol to analyze gene expression of reporters using either β-glucuronidase (GUS) or fluorescent protein (FP) tags, compatible with different dyes to stain cell walls. We detail materials, equipment, solutions, and procedures to easily implement ClearSee for the study of vascular development in Arabidopsis thaliana, but the protocol can be easily adapted to a variety of plant tissues in a wide range of plant species.

Spatial transcriptomics reveal basal sex differences in supraoptic nucleus gene expression of adult rats related to cell signaling and ribosomal pathways

BackgroundThe supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that secrete the hormones vasopressin and oxytocin. Sex differences in SON gene expression have been relatively unexplored. Our study used spatially resolved transcriptomics to visualize gene expression profiles in the SON of adult male (n = 4) and female (n = 4) Sprague-Dawley rats using Visium Spatial Gene Expression (10x Genomics).MethodsBriefly, 10-μm coronal sections (~ 4 × 4 mm) containing the SON were collected from each rat and processed using Visium slides and recommended protocols. Data were analyzed using 10x Genomics’ Space Ranger and Loupe Browser applications and other bioinformatic tools. Two unique differential expression (DE) analysis methods, Loupe Browser and DESeq2, were used.ResultsLoupe Browser DE analysis of the SON identified 116 significant differentially expressed genes (DEGs) common to both sexes (e.g., Avp and Oxt), 31 significant DEGs unique to the males, and 73 significant DEGs unique to the females. DESeq2 analysis revealed 183 significant DEGs between the two groups. Gene Ontology (GO) enrichment and pathway analyses using significant genes identified via Loupe Browser revealed GO terms and pathways related to (1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females, (2) Gi signaling/G-protein mediated events for the significant genes unique to males, and (3) potassium ion transport/voltage-gated potassium channels for the significant genes unique to females, as some examples. GO/pathway analyses using significant genes identified via DESeq2 comparing female vs. male groups revealed GO terms/pathways related to ribosomal structure/function. Ingenuity Pathway Analysis (IPA) identified additional sex differences in canonical pathways (e.g., ‘Mitochondrial Dysfunction’, ‘Oxidative Phosphorylation’) and upstream regulators (e.g., CSF3, NFKB complex, TNF, GRIN3A).ConclusionThere was little overlap in the IPA results for the two different DE methods. These results suggest sex differences in SON gene expression that are associated with cell signaling and ribosomal pathways.

Visual opsin gene expression evolution in the adaptive radiation of cichlid fishes of Lake Tanganyika.

Tuning the visual sensory system to the ambient light is essential for survival in many animal species. This is often achieved through duplication, functional diversification, and/or differential expression of visual opsin genes. Here, we examined 753 new retinal transcriptomes from 112 species of cichlid fishes from Lake Tanganyika to unravel adaptive changes in gene expression at the macro-evolutionary and ecosystem level of one of the largest vertebrate adaptive radiations. We found that, across the radiation, all seven cone opsins-but not the rhodopsin-rank among the most differentially expressed genes in the retina, together with other vision-, circadian rhythm-, and hemoglobin-related genes. We propose two visual palettes characteristic of very shallow- and deep-water living species, respectively, and show that visual system adaptations along two major ecological axes, macro-habitat and diet, occur primarily via gene expression variation in a subset of cone opsin genes.

Multi-omics atlas of combinatorial abiotic stress responses in wheat.

Field-grown crops rarely experience growth conditions in which yield can be maximized. Environmental stresses occur in combination, with advancements in crop tolerance further complicated by its polygenic nature. Strategic targeting of causal genes is required to meet future crop production needs. Here, we employed a systems biology approach in wheat (Triticum aestivum L.) to investigate physio-metabolic adjustments and transcriptome reprogramming involved in acclimations to heat, drought, salinity and all combinations therein. A significant shift in magnitude and complexity of plant response was evident across stress scenarios based on the agronomic losses, increased proline concentrations and 8.7-fold increase in unique differentially expressed transcripts (DETs) observed under the triple stress condition. Transcriptome data from all stress treatments were assembled into an online, open access eFP browser for visualizing gene expression during abiotic stress. Weighted gene co-expression network analysis revealed 152 hub genes of which 32% contained the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) transcriptional repression motif. Cross-referencing against the 31 DETs common to all stress treatments isolated TaWRKY33 as a leading candidate for greater plant tolerance to combinatorial stresses. Integration of our findings with available literature on gene functional characterization allowed us to further suggest flexible gene combinations for future adaptive gene stacking in wheat. Our approach demonstrates the strength of robust multi-omics-based data resources for gene discovery in complex environmental conditions. Accessibility of such datasets will promote cross-validation of candidate genes across studies and aid in accelerating causal gene validation for crop resiliency.

Visualizing gene expression changes in time, space, and single cells with expressyouRcell

The last decade has witnessed massive advancements in high-throughput techniques capable of producing increasingly complex gene expression datasets across time and space and at the resolution of single cells. Yet, the large volume of big data available and the complexity of experimental designs hamper an easy understanding and effective communication of the results. We present expressyouRcell, an easy-to-use R package to map the multi-dimensional variations of transcript and protein levels in dynamic cell pictographs. expressyouRcell visualizes gene expression variations as pictographic representations of cell-type thematic maps. expressyouRcell visually reduces the complexity of displaying gene expression and protein level changes across multiple measurements (time points or single-cell trajectories) by generating dynamic representations of cellular pictographs. We applied expressyouRcell to single cell, bulk RNA sequencing (RNA-seq), and proteomics datasets, demonstrating its flexibility and usability in the visualization of complex variations in gene expression. Our approach improves the standard quantitative interpretation and communication of relevant results.

A versatile genetic-encoded reporter for magnetic resonance imaging

It has been a long-cherished wish in biomedicine research to have an imaging tool to visualize gene expression, with good spatiotemporal resolution, in rodent and primate animals noninvasively and longitudinally. To this purpose, we here present a novel genetic encoded magnetic resonance imaging reporter, i.e., GEM reporter, for noninvasive visualization of cell-specific gene expression. The GEM reporter was developed through codon modification of a bacteria-originated manganese (Mn) binding protein, allowing the sequestration of endogenous Mn in local tissues. When expressed in bacteria, plant and animals, GEM reporter can robustly produce high image contrast in T1-weighted MRI without additional substrates or contrast agents. Importantly, GEM reporter can be tracked inherently by MRI in specific cells and tissues. These findings support GEM reporter as a versatile marker for deciphering gene expression spatiotemporally in living subjects.

Long-wavelength-sensitive (lws) opsin gene expression, foraging and visual communication in coral reef fishes.

Coral reef fishes are diverse in ecology and behaviour and show remarkable colour variability. Investigating the visual pigment gene (opsin) expression in these fishes makes it possible to associate their visual genotype and phenotype (spectral sensitivities) to visual tasks, such as feeding strategy or conspecific detection. By studying all major damselfish clades (Pomacentridae) and representatives from five other coral reef fish families, we show that the long-wavelength-sensitive (lws) opsin is highly expressed in algivorous and less or not expressed in zooplanktivorous species. Lws is also upregulated in species with orange/red colours (reflectance >520 nm) and expression is highest in orange/red-coloured algivores. Visual models from the perspective of a typical damselfish indicate that sensitivity to longer wavelengths does enhance the ability to detect the red to far-red component of algae and orange/red-coloured conspecifics, possibly enabling social signalling. Character state reconstructions indicate that in the early evolutionary history of damselfishes, there was no lws expression and no orange/red coloration. Omnivory was most often the dominant state. Although herbivory was sometimes dominant, zooplanktivory was never dominant. Sensitivity to long wavelength (increased lws expression) only emerged in association with algivory but never with zooplanktivory. Higher lws expression is also exploited by social signalling in orange/red, which emerged after the transition to algivory. Although the relative timing of traits may deviate by different reconstructions and alternative explanations are possible, our results are consistent with sensory bias whereby social signals evolve as a correlated response to natural selection on sensory system properties in other contexts.

FungiExp: a user-friendly database and analysis platform for exploring fungal gene expression and alternative splicing.

Fungi form a large and heterogeneous group of eukaryotic organisms with diverse ecological niches. The high importance of fungi contrasts with our limited understanding of fungal lifestyle and adaptability to environment. Over the last decade, the high-throughput sequencing technology produced tremendous RNA-sequencing (RNA-seq) data. However, there is no comprehensive database for mycologists to conveniently explore fungal gene expression and alternative splicing. Here, we have developed FungiExp, an online database including 35821 curated RNA-seq samples derived from 220 fungal species, together with gene expression and alternative splicing profiles. It allows users to query and visualize gene expression and alternative splicing in the collected RNA-seq samples. Furthermore, FungiExp contains several online analysis tools, such as differential/specific, co-expression network and cross-species gene expression conservation analysis. Through these tools, users can obtain new insights by re-analyzing public RNA-seq data or upload personal data to co-analyze with public RNA-seq data. The FungiExp is freely available at https://bioinfo.njau.edu.cn/fungiExp. Supplementary data are available at Bioinformatics online.

Identification and Localization of Cell Types in the Mouse Olfactory Bulb Using Slide-SeqV2.

Spatial transcriptomics maps RNA molecules to the location in a tissue where they are expressed. Here we document the use of Slide-SeqV2 to visualize gene expression in the mouse olfactory bulb (OB). This approach relies on spatially identified beads to locate and quantify individual transcripts. The expression profiles associated with the beads are used to identify and localize individual cell types in an unbiased manner. We demonstrate the various cell types and subtypes with distinct spatial locations in the olfactory bulb that are identified using Slide-SeqV2.

MScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans.

Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.

Whole-mount RNA in situ hybridization technique in Torenia ovules.

The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial-temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules of Torenia fournieri, which is a model species in the study of plant reproduction. T. fournieri possesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene, TfSTK3, using both methods. The expression patterns of TfSTK3 are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate, TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules of T. fournieri within one week and will benefit the field of plant reproduction in the future.

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization.

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the "serial section method" has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.

The diversity of invertebrate visual opsins spanning Protostomia, Deuterostomia, and Cnidaria

Across eumetazoans, the ability to perceive and respond to visual stimuli is largely mediated by opsins, a family of proteins belonging to the G protein-coupled receptor (GPCR) superclass. Lineage-specific gains and losses led to a striking diversity in the numbers, types, and spectral sensitivities conferred by visual opsin gene expression. Here, we review the diversity of visual opsins and differences in opsin gene expression from well-studied protostome, invertebrate deuterostome, and cnidarian groups. We discuss the functional significance of opsin expression differences and spectral tuning among lineages. In some cases, opsin evolution has been linked to the detection of relevant visual signals, including sexually selected color traits and host plant features. In other instances, variation in opsins has not been directly linked to functional or ecological differences. Overall, the array of opsin expression patterns and sensitivities across invertebrate lineages highlight the diversity of opsins in the eumetazoan ancestor and the labile nature of opsins over evolutionary time.

The temporal dynamics of the sea urchin regulome.

ABSTRACTIn this work, we used Nanostring N-counter technology, to evaluate the mRNA expression level of more than 330 regulatory genes over 34 time points covering the first three days of development of the sea urchin larvae. The hierarchical clustering of the mRNAs expression levels has identified groups corresponding to the major developmental landmarks (e.g. maternal to zygotic transition and gastrulation). Furthermore, comparison with previous experiments indicates high reproducibility of mRNA level temporal dynamics across batches. Finally, we generated an online tool to visualise gene expression during sea urchin larval development. The site can be accessed at and https://www621.lamp.le.ac.uk/nanostring_app/nanostring/.

MarpolBase Expression: A Web-Based, Comprehensive Platform for Visualization and Analysis of Transcriptomes in the Liverwort Marchantia polymorpha.

The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha and promote our understanding of the evolution of land plants.