Gene Expression Domains Research Articles

High-Throughput 3D Phenotyping of Plant Shoot Apical Meristems From Tissue-Resolution Data.

Confocal imaging is a well-established method for investigating plant phenotypes on the tissue and organ level. However, many differences are difficult to assess by visual inspection and researchers rely extensively on ad hoc manual quantification techniques and qualitative assessment. Here we present a method for quantitatively phenotyping large samples of plant tissue morphologies using triangulated isosurfaces. We successfully demonstrate the applicability of the approach using confocal imaging of aerial organs in Arabidopsis thaliana. Automatic identification of flower primordia using the surface curvature as an indication of outgrowth allows for high-throughput quantification of divergence angles and further analysis of individual flowers. We demonstrate the throughput of our method by quantifying geometric features of 1065 flower primordia from 172 plants, comparing auxin transport mutants to wild type. Additionally, we find that a paraboloid provides a simple geometric parameterisation of the shoot inflorescence domain with few parameters. We utilise parameterisation methods to provide a computational comparison of the shoot apex defined by a fluorescent reporter of the central zone marker gene CLAVATA3 with the apex defined by the paraboloid. Finally, we analyse the impact of mutations which alter mechanical properties on inflorescence dome curvature and compare the results with auxin transport mutants. Our results suggest that region-specific expression domains of genes regulating cell wall biosynthesis and local auxin transport can be important in maintaining the wildtype tissue shape. Altogether, our results indicate a general approach to parameterise and quantify plant development in 3D, which is applicable also in cases where data resolution is limited, and cell segmentation not possible. This enables researchers to address fundamental questions of plant development by quantitative phenotyping with high throughput, consistency and reproducibility.

Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration.

The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, which are defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with five out of six elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in larval hearts. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration. This article has an associated 'The people behind the papers' interview.

PaPYL9 is involved in the regulation of apricot fruit ripening through ABA signaling pathway

Abscisic acid (ABA) is a major regulator of non-climacteric fruit ripening; however, the role of ABA in the ripening of climacteric fruit is not clear. Here, as a typical climacteric fruit, apricots were used to investigate the role of ABA in fruit ripening. Based on weighted gene co-expression network analysis (WGCNA) of our previous transcriptome data, we treated ‘Danxing’ fruit with exogenous ABA and obtained ABA receptor genes, genes related to ABA biosynthesis and signal transduction, and analyzed the response of these candidate genes to exogenous ABA during fruit ripening. Subsequently, the full length of candidate PYLs genes were cloned, and their putative function were analyzed by phylogenetic analysis and protein structure domain analysis. And then the function of one candidate gene PaPYL9 was verified by using transgenic tomato. Furthermore, the response genes in transgenic tomato were screened by transcriptome sequencing, and ultimately the related regulatory network was proposed. The results showed that the injection of exogenous 1.89 mmol·L-1 ABA remarkably promoted fruit coloration, and increased the color index for red grapes (CIRG) and the total soluble solids (TSS) content, but significantly decreased the firmness and titratable acid (TA) content (p < 0.01). Nordihydroguaiaretic acid (NDGA), the inhibitor of ABA, appeared to have the converse role in TA, TSS, CIRG and firmness, during the ripening process. One NCED (9-cis-epoxycarotenoiddioxygenase) and five ABA receptor genes related to signal transduction were mined from the transcriptome data of apricot fruit through WGCNA. Compared with the control, the expression levels of NCED1, PYL9 (PYR/PYL/RCAR), SnRK2 (SUCROSE NON-FERMENTING1 (SNF1)-RELATED PROTEIN KINASE 2S), and ABF2 (ABRE-binding bZIP transcription) were induced dramatically by ABA treatment (p < 0.01), while NDGA treatment significantly inhibited their expression. Based on gene expression and protein domain analysis, we inferred that PaPYL9 is putatively involved in apricot fruit ripening. Overexpression of PaPYL9 in Micro-TOM tomatoes resulted in the promotion of early ripening. Simultaneously, the expression levels of genes related ethylene biosynthesis, chlorophyll degradation, fruit softening, flavor formation, pigment synthesis, and metabolism were all significantly induced in overexpression of PaPYL9 tomatoes. This indicates the central role of ABA in climacteric fruit ripening. A regulatory network was tentatively proposed, laying the foundation to unveil the molecular mechanism of the regulatory role of PaPYL9 in fruit ripening.

Evolution of inwardly rectifying potassium channels and their gene expression in zebrafish embryos.

Inwardly rectifying potassium channels are essential for normal potassium homeostasis, maintaining the cellular resting membrane potential, and regulating electrolyte transportation. Mutations in Kir channels have been known to cause debilitating diseases ranging from neurological abnormalities to renal and cardiac failures. Many efforts have been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and functions during embryonic development remain largely unknown. Using zebrafish as a model, we identified and renamed 31 kir genes. We also analyzed Kir gene evolution by phylogenetic and syntenic analyses. Our data indicated that the four subtypes of the Kir genes might have already evolved out in chordates. These vertebrate Kir genes most likely resulted from both whole-genome duplications and tandem duplications. In addition, we examined zebrafish kir gene expression during early embryogenesis. Each subgroup's genes showed similar but distinct gene expression domains. The gene expression of ohnologous genes from teleost-specific whole-genome duplication indicated subfunctionalization. Varied temporal gene expression domains suggest that Kir channels may be needed for embryonic patterning or regulation. Our phylogenetic and developmental analyses of Kir channels shed light on their evolutionary history and potential functions during embryogenesis related to congenital diseases and human channelopathies.

Multiple morphogens and rapid elongation promote segmental patterning during development.

The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development.

Implications of diffusion and time-varying morphogen gradients for the dynamic positioning and precision of bistable gene expression boundaries.

The earliest models for how morphogen gradients guide embryonic patterning failed to account for experimental observations of temporal refinement in gene expression domains. Following theoretical and experimental work in this area, dynamic positional information has emerged as a conceptual framework to discuss how cells process spatiotemporal inputs into downstream patterns. Here, we show that diffusion determines the mathematical means by which bistable gene expression boundaries shift over time, and therefore how cells interpret positional information conferred from morphogen concentration. First, we introduce a metric for assessing reproducibility in boundary placement or precision in systems where gene products do not diffuse, but where morphogen concentrations are permitted to change in time. We show that the dynamics of the gradient affect the sensitivity of the final pattern to variation in initial conditions, with slower gradients reducing the sensitivity. Second, we allow gene products to diffuse and consider gene expression boundaries as propagating wavefronts with velocity modulated by local morphogen concentration. We harness this perspective to approximate a PDE model as an ODE that captures the position of the boundary in time, and demonstrate the approach with a preexisting model for Hunchback patterning in fruit fly embryos. We then propose a design that employs antiparallel morphogen gradients to achieve accurate boundary placement that is robust to scaling. Throughout our work we draw attention to tradeoffs among initial conditions, boundary positioning, and the relative timescales of network and gradient evolution. We conclude by suggesting that mathematical theory should serve to clarify not just our quantitative, but also our intuitive understanding of patterning processes.

Novel cell types and developmental lineages revealed by single-cell RNA-seq analysis of the mouse crista ampullaris.

This study provides transcriptomic characterization of the cells of the crista ampullaris, sensory structures at the base of the semicircular canals that are critical for vestibular function. We performed single-cell RNA-seq on ampullae microdissected from E16, E18, P3, and P7 mice. Cluster analysis identified the hair cells, support cells and glia of the crista as well as dark cells and other nonsensory epithelial cells of the ampulla, mesenchymal cells, vascular cells, macrophages, and melanocytes. Cluster-specific expression of genes predicted their spatially restricted domains of gene expression in the crista and ampulla. Analysis of cellular proportions across developmental time showed dynamics in cellular composition. The new cell types revealed by single-cell RNA-seq could be important for understanding crista function and the markers identified in this study will enable the examination of their dynamics during development and disease.

Three-dimensional multi-gene expression maps reveal cell fate changes associated with laterality reversal of zebrafish habenula.

The conserved bilateral habenular nuclei (HA) in vertebrate diencephalon develop into compartmentalized structures containing neurons derived from different cell lineages. Despite extensive studies demonstrated that zebrafish larval HA display distinct left-right (L-R) asymmetry in gene expression and connectivity, the spatial gene expression domains were mainly obtained from two-dimensional (2D) snapshots of colorimetric RNA in situ hybridization staining which could not properly reflect different HA neuronal lineages constructed in three-dimension (3D). Combing the tyramide-based fluorescent mRNA in situ hybridization, confocal microscopy and customized imaging processing procedures, we have created spatial distribution maps of four genes for 4-day-old zebrafish and in sibling fish whose L-R asymmetry was spontaneously reversed. 3D volumetric analyses showed that ratios of cpd2, lov, ron, and nrp1a expression in L-R reversed HA were reversed according to the parapineal positions. However, the quantitative changes of gene expression in reversed larval brains do not mirror the gene expression level in the obverse larval brains. There were a total 87.78% increase in lov+ nrp1a+ and a total 12.45% decrease in lov+ ron+ double-positive neurons when the L-R asymmetry of HA was reversed. Thus, our volumetric analyses of the 3D maps indicate that changes of HA neuronal cell fates are associated with the reversal of HA laterality. These changes likely account for the behavior changes associated with HA laterality alterations.

Precision of tissue patterning is controlled by dynamical properties of gene regulatory networks.

ABSTRACTDuring development, gene regulatory networks allocate cell fates by partitioning tissues into spatially organised domains of gene expression. How the sharp boundaries that delineate these gene expression patterns arise, despite the stochasticity associated with gene regulation, is poorly understood. We show, in the vertebrate neural tube, using perturbations of coding and regulatory regions, that the structure of the regulatory network contributes to boundary precision. This is achieved, not by reducing noise in individual genes, but by the configuration of the network modulating the ability of stochastic fluctuations to initiate gene expression changes. We use a computational screen to identify network properties that influence boundary precision, revealing two dynamical mechanisms by which small gene circuits attenuate the effect of noise in order to increase patterning precision. These results highlight design principles of gene regulatory networks that produce precise patterns of gene expression.

Non-collinear Hox gene expression in bivalves and the evolution of morphological novelties in mollusks

Hox genes are key developmental regulators that are involved in establishing morphological features during animal ontogeny. They are commonly expressed along the anterior–posterior axis in a staggered, or collinear, fashion. In mollusks, the repertoire of body plans is widely diverse and current data suggest their involvement during development of landmark morphological traits in Conchifera, one of the two major lineages that comprises those taxa that originated from a uni-shelled ancestor (Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda, Bivalvia). For most clades, and bivalves in particular, data on Hox gene expression throughout ontogeny are scarce. We thus investigated Hox expression during development of the quagga mussel, Dreissena rostriformis, to elucidate to which degree they might contribute to specific phenotypic traits as in other conchiferans. The Hox/ParaHox complement of Mollusca typically comprises 14 genes, 13 of which are present in bivalve genomes including Dreissena. We describe here expression of 9 Hox genes and the ParaHox gene Xlox during Dreissena development. Hox expression in Dreissena is first detected in the gastrula stage with widely overlapping expression domains of most genes. In the trochophore stage, Hox gene expression shifts towards more compact, largely mesodermal domains. Only few of these domains can be assigned to specific developing morphological structures such as Hox1 in the shell field and Xlox in the hindgut. We did not find traces of spatial or temporal staggered expression of Hox genes in Dreissena. Our data support the notion that Hox gene expression has been coopted independently, and to varying degrees, into lineage-specific structures in the respective conchiferan clades. The non-collinear mode of Hox expression in Dreissena might be a result of the low degree of body plan regionalization along the bivalve anterior–posterior axis as exemplified by the lack of key morphological traits such as a distinct head, cephalic tentacles, radula apparatus, and a simplified central nervous system.

The BMP signaling gradient is interpreted through concentration thresholds in dorsal-ventral axial patterning.

Bone Morphogenetic Protein (BMP) patterns the dorsal–ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.

Coevolution of enamel, ganoin, enameloid, and their matrix SCPP genes in osteichthyans

SummaryWe resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes.

Gastruloids: Embryonic Organoids from Mouse Embryonic Stem Cells to Study Patterning and Development in Early Mammalian Embryos.

Gastruloids are embryonic organoids made from small, defined numbers of mouse embryonic stem cells (mESCs) aggregated in suspension culture, which over time form 3D structures that mimic many of the features of early mammalian development. Unlike embryoid bodies that are usually disorganized when grown over several days, gastruloids display distinct, well-organized gene expression domains demarcating the emergence of the three body axes, anteroposterior axial elongation, and implementation of collinear Hox transcriptional patterns over 5-7days of culture. As such gastruloids represent a useful experimental system that is complementary to in vivo approaches in studying early developmental patterning mechanisms regulating the acquisition of cell fates. In this protocol, we describe the most recent method for generating gastruloids with high reproducibility, and provide a comprehensive list of possible challenges as well as steps for protocol optimization.

A pitfall for machine learning methods aiming to predict across cell types

Machine learning models that predict genomic activity are most useful when they make accurate predictions across cell types. Here, we show that when the training and test sets contain the same genomic loci, the resulting model may falsely appear to perform well by effectively memorizing the average activity associated with each locus across the training cell types. We demonstrate this phenomenon in the context of predicting gene expression and chromatin domain boundaries, and we suggest methods to diagnose and avoid the pitfall. We anticipate that, as more data becomes available, future projects will increasingly risk suffering from this issue.

Interpretation of morphogen gradients by a synthetic bistable circuit

During development, cells gain positional information through the interpretation of dynamic morphogen gradients. A proposed mechanism for interpreting opposing morphogen gradients is mutual inhibition of downstream transcription factors, but isolating the role of this specific motif within a natural network remains a challenge. Here, we engineer a synthetic morphogen-induced mutual inhibition circuit in E. coli populations and show that mutual inhibition alone is sufficient to produce stable domains of gene expression in response to dynamic morphogen gradients, provided the spatial average of the morphogens falls within the region of bistability at the single cell level. When we add sender devices, the resulting patterning circuit produces theoretically predicted self-organised gene expression domains in response to a single gradient. We develop computational models of our synthetic circuits parameterised to timecourse fluorescence data, providing both a theoretical and experimental framework for engineering morphogen-induced spatial patterning in cell populations.

ATAC-Seq for Assaying Chromatin Accessibility Protocol Using Echinoderm Embryos.

Cis-regulatory elements (CREs) and transcription factors (TFs) associated with them determine temporal and spatial domains of gene expression. Therefore, identification of these CREs and TFs is crucial to elucidating transcriptional programs across taxa. With chromatin accessibility facilitating transcription factor access to DNA, the identification of regions of open chromatin sheds light both on the function of the regulatory elements and their evolution, thus allowing the recognition of potential CREs. Buenrostro and colleagues have developed a novel method for exploring chromatin accessibility: assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), which can be used for the purpose of identifying putative CREs. This method was shown to have considerable advantages when compared to traditional methods such as sequence conservation analyses or functional assays. Here we present the adaptation of the ATAC-seq method to echinoderm species and discuss how it can be used for CRE discovery.

Extensive fragmentation and re-organization of transcription in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is the prototype of autoimmune diseases, characterized by extensive gene expression perturbations in peripheral blood immune cells. Circumstantial evidence suggests that these perturbations may be due to altered epigenetic profiles and chromatin accessibility but the relationship between transcriptional deregulation and genome organization remains largely unstudied. In this work we propose a genomic approach that leverages patterns of gene coexpression from genome-wide transcriptome profiles in order to identify statistically robust Domains of Co-ordinated gene Expression (DCEs). Application of this method on a large transcriptome profiling dataset of 148 SLE patients and 52 healthy individuals enabled the identification of significant disease-associated alterations in gene co-regulation patterns, which also correlate with SLE activity status. Low disease activity patient genomes are characterized by extensive fragmentation leading to overall fewer DCEs of smaller size. High disease activity genomes display extensive redistribution of co-expression domains with expanded and newly-appearing (emerged) DCEs. The dynamics of domain fragmentation and redistribution are associated with SLE clinical endophenotypes, with genes of the interferon pathway being highly enriched in DCEs that become disrupted and with functions associated to more generalized symptoms, being located in domains that emerge anew in high disease activity genomes. Our results suggest strong links between the SLE phenotype and the underlying genome structure and underline an important role for genome organization in shaping gene expression in SLE.

Sox17 and β-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

Hypermobile Ehlers-Danlos syndromes: Complex phenotypes, challenging diagnoses, and poorly understood causes.

The Ehlers-Danlos syndromes (EDS) are a group of heritable, connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. There is phenotypic and genetic variation among the 13 subtypes. The initial genetic findings on EDS were related to alterations in fibrillar collagen, but the elucidation of the molecular basis of many of the subtypes revealed several genes not involved in collagen biosynthesis or structure. However, the genetic basis of the hypermobile type of EDS (hEDS) is still unknown. hEDS is the most common type of EDS and involves generalized joint hypermobility, musculoskeletal manifestations, and mild skin involvement along with the presence of several comorbid conditions. Variability in the spectrum and severity of symptoms and progression of patient phenotype likely depend on age, gender, lifestyle, and expression domains of the EDS genes during development and postnatal life. In this review, we summarize the current molecular, genetic, epidemiologic, and pathogenetic findings related to EDS with a focus on the hypermobile type.

A novel scheme for essential protein discovery based on multi-source biological information

Mining essential protein is crucial for discovering the process of cellular organization and viability. At present, there are many computational methods for essential proteins detecting. However, these existing methods only focus on the topological information of the networks and ignore the biological information of proteins, which lead to low accuracy of essential protein identification. Therefore, this paper presents a new essential proteins prediction strategy, called DEP-MSB which integrates a variety of biological information including gene expression profiles, GO annotations, and Domain interaction strength. In order to evaluate the performance of DEP-MSB, we conduct a series of experiments on the yeast PPI network and the experimental results have shown that the proposed algorithm DEP-MSB is more superior to the other existing traditional methods and has obviously improvement in prediction accuracy.