scholarly journals A Transcriptional Lineage of the Early C. elegans Embryo

2016 ◽  
Author(s):  
Sophia C. Tintori ◽  
Erin Osborne Nishimura ◽  
Patrick Golden ◽  
Jason D. Lieb ◽  
Bob Goldstein
Keyword(s):  
Single Cell ◽  
Data Visualization ◽  
Cell Lineage ◽  
List Type ◽  
Visualization Tool ◽  
Rna Seq ◽  
Online Data ◽  
Cell Stage ◽  
C Elegans ◽  
Zygotic Genome

HIGHLIGHTS‒RNA-seq on each cell of the early C. elegans embryo complements the known lineage‒We measured the zygotic activation specific to each unique cell of the embryo‒We identified genes that are functionally redundant and critical for development‒We created an interactive online data visualization tool for exploring the dataeTOC BLURBC. elegans is a powerful model for development, with an invariant and completely described cell lineage. To enrich this resource, we performed single-cell RNA-seq on each cell of the embryo through the 16-cell stage. Zygotic genome activation is differential between cell types. We identified hundreds of candidates for partially redundant genes, and verified one such set as critical for development. We created an interactive online data visualization tool to invite others to explore our dataset.SUMMARYDuring embryonic development, cells must establish fates, morphologies and behaviors in coordination with one another to form a functional body. A prevalent hypothesis for how this coordination is achieved is that each cell’s fate and behavior is determined by a defined mixture of RNAs. Only recently has it become possible to measure the full suite of transcripts in a single cell. Here we quantify the abundance of every mRNA transcript in each cell of the C. elegans embryo up to the 16-cell stage. We describe spatially dynamic expression, quantify cell-specific differential activation of the zygotic genome, and identify critical developmental genes previously unappreciated because of their partial redundancy. We present an interactive data visualization tool that allows broad access to our dataset. This genome-wide single-cell map of mRNA abundance, alongside the well-studied life history and fates of each cell, describes at a cellular resolution the mRNA landscape that guides development.

Formation of the egg-laying system inPristionchus pacificusrequires complex interactions between gonadal, mesodermal and epidermal tissues and does not rely on single cell inductions

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3395-3404
Author(s):  
Benno Jungblut ◽  
André Pires-daSilva ◽  
Ralf J. Sommer
Keyword(s):  
Single Cell ◽  
Lateral Inhibition ◽  
Cell Lineage ◽  
Egg Laying ◽  
C Elegans ◽  
Complex Interactions ◽  
Organ Systems ◽  
Vulval Precursor Cells ◽  
Sex Myoblasts ◽  
Multiple Cells

The invariant cell lineage of nematodes allows the formation of organ systems, like the egg-laying system, to be studied at a single cell level. The Caenorhabditis elegans egg-laying system is made up of the vulva, the mesodermal gonad and muscles and several neurons. The gonad plays a central role in patterning the underlying ectoderm to form the vulva and guiding the migration of the sex myoblasts to their final position. In Pristionchus pacificus, the egg-laying system is homologous to C. elegans, but comparative studies revealed several differences at the cellular and molecular levels during vulval formation. For example, the mesoblast M participates in lateral inhibition, a process that influences the fate of two vulval precursor cells. Here, we describe the M lineage in Pristionchus and show that both the dorsal and ventral M sublineages are involved in lateral inhibition. Mutations in the homeotic gene Ppa-mab-5 cause severe misspecification of the M lineage, resembling more the C. elegans Twist than the mab-5 phenotype. Ectopic differentiation of P8.p in Ppa-mab-5 results from at least two separate interactions between M and P8.p. Thus, interactions among the Pristionchus egg-laying system are complex, involving multiple cells of different tissues occurring over a distance.

An analysis of the response to gut induction in the C. elegans embryo

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1227-1236 ◽  
Author(s):  
B. Goldstein
Keyword(s):  
Body Wall ◽  
Cell Lineage ◽  
Muscle Cells ◽  
The Other ◽  
Cell Stage ◽  
Pharyngeal Muscle ◽  
Cell Cycles ◽  
Sister Cell ◽  
C Elegans ◽  
Cell Diversity

Establishment of the gut founder cell (E) in C. elegans involves an interaction between the P2 and the EMS cell at the four cell stage. Here I show that the fate of only one daughter of EMS, the E cell, is affected by this induction. In the absence of the P2-EMS interaction, both E and its sister cell, MS, produce pharyngeal muscle cells and body wall muscle cells, much as MS normally does. By cell manipulations and inhibitor studies, I show first that EMS loses the competence to respond before it divides even once, but P2 presents an inducing signal for at least three cell cycles. Second, induction on one side of the EMS cell usually blocks the other side from responding to a second P2-derived signal. Third, microfilaments and microtubules may be required near the time of the interaction for subsequent gut differentiation. Lastly, cell manipulations in pie-1 mutant embryos, in which the P2 cell is transformed to an EMS-like fate and produces a gut cell lineage, revealed that gut fate is segregated to one of P2's daughters cell-autonomously. The results contrast with previous results from similar experiments on the response to other inductions, and suggest that this induction may generate cell diversity by a different mechanism.

scholarly journals Microinjection into the Caenorhabditis elegans embryo using an uncoated glass needle enables cell lineage visualization and reveals cell-non-autonomous adhesion control

2018 ◽  
Author(s):  
Yohei Kikuchi ◽  
Akatsuki Kimura
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Biology ◽  
Cell Lineage ◽  
Special Equipment ◽  
Cell Stage ◽  
C Elegans ◽  
Carbon Coated ◽  
A Cell ◽  
Glass Needle ◽  
Time Specific

AbstractMicroinjection is a useful method in cell biology, with which exogenous substances are introduced into a cell in a location- and time-specific manner. The Caenorhabditis elegans embryo is an important model system for cell and developmental biology. Applying microinjection to the C. elegans embryo had been difficult due to the rigid eggshell surrounding the embryo. In 2013, microinjection method using a carbon-coated quartz needle for the C. elegans embryo was reported. To prepare the needle, unfortunately, special equipment is required and thus a limited number of researchers can use this method. In this study, we established a method for the microinjection of drugs, dyes, and microbeads into the C. elegans embryo using an uncoated glass needle that can be produced in a general laboratory. This method enabled us to easily detect cell lineage up to adult stages by injecting a fluorescent dye into a blastomere. We also found a cell-non-autonomous control mechanism of cell adhesion; specifically, the injection of an actin inhibitor into one cell at the 2-cell stage enhanced adhesion between daughter cells of the other cell. Our microinjection method is expected to be used for broad studies and could facilitate various discoveries using C. elegans.

scholarly journals Differential Expression Gene Explorer (DrEdGE): A tool for generating interactive online data visualizations for exploration of quantitative transcript abundance datasets

2019 ◽  
Author(s):  
Sophia C. Tintori ◽  
Patrick Golden ◽  
Bob Goldstein
Keyword(s):  
Data Sharing ◽  
Transcript Abundance ◽  
Rna Seq ◽  
Online Data ◽  
Web Based ◽  
Neuronal Tissue ◽  
C Elegans ◽  
Differential Expression Gene ◽  
Public Data ◽  
Data Visualizations

AbstractAs the scientific community becomes increasingly interested in data sharing, there is a growing need for tools that facilitate the querying of public data. Mining of RNA-seq datasets, for example, has value to many biomedical researchers, yet is often effectively inaccessible to non-genomicist experts, even when the raw data are available. Here we present DrEdGE (dredge.bio.unc.edu), a free Web-based tool that facilitates data sharing between genomicists and their colleagues. The DrEdGE software guides genomicists through easily creating interactive online data visualizations, which colleagues can then explore and query according to their own conditions to discover genes, samples, or patterns of interest. We demonstrate DrEdGE’s features with three example websites we generated from publicly available datasets—human neuronal tissue, mouse embryonic tissue, and a C. elegans embryonic series. DrEdGE increases the utility of large genomics datasets by removing the technical obstacles that prevent interested parties from exploring the data independently.

scholarly journals Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

2017 ◽  
Author(s):  
Junyue Cao ◽  
Jonathan S. Packer ◽  
Vijay Ramani ◽  
Darren A. Cusanovich ◽  
Chau Huynh ◽  
...  
Keyword(s):  
Single Cell ◽  
Expression Profiles ◽  
Single Cells ◽  
Transcriptional Profiling ◽  
Cost Effective ◽  
Cell Types ◽  
Cell Capture ◽  
Rna Seq ◽  
Major Step ◽  
C Elegans

AbstractConventional methods for profiling the molecular content of biological samples fail to resolve heterogeneity that is present at the level of single cells. In the past few years, single cell RNA sequencing has emerged as a powerful strategy for overcoming this challenge. However, its adoption has been limited by a paucity of methods that are at once simple to implement and cost effective to scale massively. Here, we describe a combinatorial indexing strategy to profile the transcriptomes of large numbers of single cells or single nuclei without requiring the physical isolation of each cell (Single cell Combinatorial Indexing RNA-seq or sci-RNA-seq). We show that sci-RNA-seq can be used to efficiently profile the transcriptomes of tens-of-thousands of single cells per experiment, and demonstrate that we can stratify cell types from these data. Key advantages of sci-RNA-seq over contemporary alternatives such as droplet-based single cell RNA-seq include sublinear cost scaling, a reliance on widely available reagents and equipment, the ability to concurrently process many samples within a single workflow, compatibility with methanol fixation of cells, cell capture based on DNA content rather than cell size, and the flexibility to profile either cells or nuclei. As a demonstration of sci-RNA-seq, we profile the transcriptomes of 42,035 single cells from C. elegans at the L2 stage, effectively 50-fold “shotgun cellular coverage” of the somatic cell composition of this organism at this stage. We identify 27 distinct cell types, including rare cell types such as the two distal tip cells of the developing gonad, estimate consensus expression profiles and define cell-type specific and selective genes. Given that C. elegans is the only organism with a fully mapped cellular lineage, these data represent a rich resource for future methods aimed at defining cell types and states. They will advance our understanding of developmental biology, and constitute a major step towards a comprehensive, single-cell molecular atlas of a whole animal.

scholarly journals Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes

2020 ◽  
Author(s):  
Timothy J. Durham ◽  
Riza M. Daza ◽  
Louis Gevirtzman ◽  
Darren A. Cusanovich ◽  
William Stafford Noble ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Expression Patterns ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Gene Expression Patterns ◽  
Rna Seq ◽  
Cell Type ◽  
Tissue Specific ◽  
C Elegans

AbstractRecently developed single cell technologies allow researchers to characterize cell states at ever greater resolution and scale. C. elegans is a particularly tractable system for studying development, and recent single cell RNA-seq studies characterized the gene expression patterns for nearly every cell type in the embryo and at the second larval stage (L2). Gene expression patterns are useful for learning about gene function and give insight into the biochemical state of different cell types; however, in order to understand these cell types, we must also determine how these gene expression levels are regulated. We present the first single cell ATAC-seq study in C. elegans. We collected data in L2 larvae to match the available single cell RNA-seq data set, and we identify tissue-specific chromatin accessibility patterns that align well with existing data, including the L2 single cell RNA-seq results. Using a novel implementation of the latent Dirichlet allocation algorithm, we leverage the single-cell resolution of the sci-ATAC-seq data to identify accessible loci at the level of individual cell types, providing new maps of putative cell type-specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation in the worm.

scholarly journals Normalization of single-cell RNA-seq counts by log(x+1)* or log(1+x)*

2020 ◽  
Author(s):  
A. Sina Booeshaghi ◽  
Lior Pachter
Keyword(s):  
Single Cell ◽  
List Type ◽  
Rna Seq ◽  
Cell Type Specificity ◽  
Alternative Approach ◽  
Key Points ◽  
Highly Expressed Genes ◽  
Low Levels ◽  
Resolution Cell ◽  
Host Genes

AbstractSingle-cell RNA-seq technologies have been successfully employed over the past decade to generate many high resolution cell atlases. These have proved invaluable in recent efforts aimed at understanding the cell type specificity of host genes involved in SARS-CoV-2 infections. While single-cell atlases are based on well-sampled highly-expressed genes, many of the genes of interest for understanding SARS-CoV-2 can be expressed at very low levels. Common assumptions underlying standard single-cell analyses don’t hold when examining low-expressed genes, with the result that standard workflows can produce misleading results.Key PointsLowly expressed genes in single-cell RNA-seq can be easliy misanalyzed.log(1+x) count normalization introduces errors for lowly expressed genesThe average log(1+x) expression differs considerably from log(x) when x is smallAn alternative approach is to use the fraction of cells with non-zero expression

205 RNA-SEq PROFILING OF BOVINE PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 251
Author(s):  
S. Krebs ◽  
A. Graf ◽  
Z. Valeri ◽  
H. Blum ◽  
E. Wolf
Keyword(s):  
Differentially Expressed Genes ◽  
Developmental Stages ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Cell Stage ◽  
Total Rna ◽  
Genome Activation ◽  
Zygotic Genome

In order to provide a comprehensive view of the transcriptome changes during the earliest stages of bovine development, we sequenced the total RNA content of bovine oocytes, 4-cell, 8-cell, and 16-cell embryos and the inner cell mass and trophoblast envelope of expanded blastocysts on the Illumina Genome Analyzer IIx. For each experiment pools of in vitro matured oocytes from the German Simmental cows were fertilized by sperm of a single bull, and 10 oocytes or embryos per developmental stage were collected to generate total RNA pools used for sequencing. Synthesis of cDNA was initiated directly in the cell lysate in order to avoid any losses during RNA preparation and was random primed in order to capture all RNA species. Amplified cDNA and unstranded sequencing libraries were prepared using kits from Nugen (Ovation RNA-Seq, Nugen, San Carlos, CA, USA). Biological replicates were generated by inseminating the oocytes with sperm from the distant breeds Jersey (n = 3) and Brahman (n = 3). This cross-breeding design allowed tracking of single sequencing reads back to the maternal or paternal genome, where breed-specific SNP are present in the expressed transcripts. The analysis of this dataset resulted in monitoring of zygotic genome activation and parent-specific expression for single transcripts, a catalogue of splicing isoforms, novel transcripts, and non-coding RNAs and differentially expressed genes between the single developmental stages. Using the program DESEqn, 2784 genes showed differential expression between any of the stages at a false discovery rate of 1%. Specifically, we found 200 genes differentially expressed between immature and matured oocytes, 209 genes between matured oocytes and 4-cell embryos, 580 genes between the 4-cell and 8-cell stage, 567 genes between the 8-cell and 16-cell stage, 987 genes between the 16-cell stage and the inner cell mass, and 1569 genes between the 16-cell and the trophoblast. Functional analysis revealed stage-specific functions of the differentially expressed genes. In summary, by fully exploiting the single-nucleotide resolution of the RNA-Seq method, this dataset provides an invaluable resource for the study of zygotic genome activation, imprinting, transcript annotation, and gene expression in the earliest developmental stages of bovine embryos.

scholarly journals Cardelino: Integrating whole exomes and single-cell transcriptomes to reveal phenotypic impact of somatic variants

2018 ◽  
Author(s):  
Davis J. McCarthy ◽  
Raghd Rostom ◽  
Yuanhua Huang ◽  
Daniel J. Kunz ◽  
Petr Danecek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Dermal Fibroblast ◽  
Human Fibroblasts ◽  
Human Dermal Fibroblast ◽  
Computational Method ◽  
Neutral Evolution ◽  
List Type ◽  
Rna Seq ◽  
Sequencing Data

AbstractDecoding the clonal substructures of somatic tissues sheds light on cell growth, development and differentiation in health, ageing and disease. DNA-sequencing, either using bulk or using single-cell assays, has enabled the reconstruction of clonal trees from frequency and co-occurrence patterns of somatic variants. However, approaches to systematically characterize phenotypic and functional variations between individual clones are not established. Here we present cardelino (https://github.com/PMBio/cardelino), a computational method for inferring the clone of origin of individual cells that have been assayed using single-cell RNA-seq (scRNA-seq). After validating our model using simulations, we apply cardelino to matched scRNA-seq and exome sequencing data from 32 human dermal fibroblast lines, identifying hundreds of differentially expressed genes between cells from different somatic clones. These genes are frequently enriched for cell cycle and proliferation pathways, indicating a key role for cell division genes in non-neutral somatic evolution.Key findingsA novel approach for integrating DNA-seq and single-cell RNA-seq data to reconstruct clonal substructure for single-cell transcriptomes.Evidence for non-neutral evolution of clonal populations in human fibroblasts.Proliferation and cell cycle pathways are commonly distorted in mutated clonal populations.

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