Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations ...and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing.
As the applications and accessibility of single-cell sequencing approaches expand, a number of tenets regarding experimental design and data analysis are important to keep in mind.
Cellular decision making is the process whereby cells assume different, functionally important and heritable fates without an associated genetic or environmental difference. Such stochastic cell fate ...decisions generate nongenetic cellular diversity, which may be critical for metazoan development as well as optimized microbial resource utilization and survival in a fluctuating, frequently stressful environment. Here, we review several examples of cellular decision making from viruses, bacteria, yeast, lower metazoans, and mammals, highlighting the role of regulatory network structure and molecular noise. We propose that cellular decision making is one of at least three key processes underlying development at various scales of biological organization.
Single-cell analyses have provided invaluable insights into studying heterogenity, signaling, and stochastic gene expression. Recent technological advances now open the door to genome-wide ...single-cell studies.
Decades ago, two nonoverlapping crypt stem cell populations were proposed: Leblond’s Crypt Base Columnar (CBC) cell and Potten’s +4 cell. The identification of CBC markers including Lgr5 has ...confirmed Leblond’s predictions that CBC cells are anatomically distinct, long-lived stem cells that permanently cycle. While Potten originally described +4 cells as proliferative and unusually radiation-sensitive, recent efforts to identify +4 stem cells have focused on the identification of cells that are quiescent and radiation-resistant. Here, we describe commonalities and discrepancies between the individual studies and discuss challenges of marker-based lineage tracing.
The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that ...surrounds a fluid-filled cavity sheltering the embryonic cells
. From mouse blastocysts, it is possible to derive both trophoblast
and embryonic stem-cell lines
, which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.
Adult mitotic tissues like the intestine, skin, and blood undergo constant turnover throughout the life of an organism. Knowing the identity of the stem cell is crucial to understanding tissue ...homeostasis and its aberrations upon disease. Here we present a computational method for the derivation of a lineage tree from single-cell transcriptome data. By exploiting the tree topology and the transcriptome composition, we establish StemID, an algorithm for identifying stem cells among all detectable cell types within a population. We demonstrate that StemID recovers two known adult stem cell populations, Lgr5+ cells in the small intestine and hematopoietic stem cells in the bone marrow. We apply StemID to predict candidate multipotent cell populations in the human pancreas, a tissue with largely uncharacterized turnover dynamics. We hope that StemID will accelerate the search for novel stem cells by providing concrete markers for biological follow-up and validation.
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•StemID infers the lineage tree and identifies stem cells from single-cell mRNA-seq data•Direct links of stem cells to distinct sub-types reflect transcriptome plasticity•The permissive stem cell transcriptome is characterized by high entropy•StemID infers candidate multipotent cell populations in the human pancreas
Grün et al. developed an algorithm, StemID, for the derivation of cell lineage trees and identification of stem cells from single-cell mRNA sequencing data. StemID successfully recovered known adult stem cell populations from the small intestine and bone marrow and was then used to predict a novel multipotent cell population in the human pancreas.
Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry ...among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.
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•Wild-type yeast and mouse cells build ribosomes with different protein composition•The stoichiometry among ribosomal proteins (RP) correlates to growth rate•RP stoichiometry depends on the number of ribosomes bound per mRNA•RP stoichiometry depends on the growth conditions
Indirect evidence gathered over decades has suggested the existence of ribosomes with distinct protein composition and translational specificity in unperturbed wild-type cells. Slavov and colleagues report direct evidence for such ribosome heterogeneity in yeast and mouse stem cells and correlative evidence for its physiological impact on cell growth.
MicroRNA control of protein expression noise Schmiedel, Jörn M.; Klemm, Sandy L.; Zheng, Yannan ...
Science (American Association for the Advancement of Science),
04/2015, Volume:
348, Issue:
6230
Journal Article
Peer reviewed
MicroRNAs (miRNAs) repress the expression of many genes in metazoans by accelerating messenger RNA degradation and inhibiting translation, thereby reducing the level of protein. However, miRNAs only ...slightly reduce the mean expression of most targeted proteins, leading to speculation about their role in the variability, or noise, of protein expression. We used mathematical modeling and single-cell reporter assays to show that miRNAs, in conjunction with increased transcription, decrease protein expression noise for lowly expressed genes but increase noise for highly expressed genes. Genes that are regulated by multiple miRNAs show more-pronounced noise reduction. We estimate that hundreds of (lowly expressed) genes in mouse embryonic stem cells have reduced noise due to substantial miRNA regulation. Our findings suggest that miRNAs confer precision to protein expression and thus offer plausible explanations for the commonly observed combinatorial targeting of endogenous genes by multiple miRNAs, as well as the preferential targeting of lowly expressed genes.
Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited ...for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.
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•A method for spatially resolved transcriptomics in 3D using standard lab equipment•Genome-wide resource of 3D zebrafish expression patterns at three embryonic stages•Identification of expression patterns, including genes with unknown function•Search for expression patterns without the need for annotation of anatomical terms
An approach that combines histology with RNA sequencing and mathematical modeling allows for the construction of a genome-wide 3D atlas of gene expression in the developing zebrafish embryo.
Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT) has been reported to alter metastasis by changing migration and stem cell capacity of ...tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells may adapt transient and reversible states. Here, we have tested the existence and role of epithelial-mesenchymal plasticity in metastasis of mammary tumors without artificially modifying EMT regulators. In these tumors, we found by intravital microscopy that the motile tumor cells have undergone EMT, while their epithelial counterparts were not migratory. Moreover, we found that epithelial-mesenchymal plasticity renders any EMT-induced stemness differences, as reported previously, irrelevant for metastatic outgrowth, because mesenchymal cells that arrive at secondary sites convert to the epithelial state within one or two divisions, thereby obtaining the same stem cell potential as their arrived epithelial counterparts. We conclude that epithelial-mesenchymal plasticity supports migration but additionally eliminates stemness-enhanced metastatic outgrowth differences.
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•Direct evidence of EMT obtained in unperturbed breast tumors by real-time visualization•EMT exists in breast tumors without experimentally altering EMT inducers•Tumor cells that underwent EMT are the migratory cells within a tumor•Outgrowth potential differences between states are irrelevant due to plasticity
Beerling et al. identified a previously undetectable pool of cells in epithelial breast tumors that have undergone EMT without experimental induction. These cells are motile when disseminating and revert back to the epithelial state upon metastatic outgrowth. This epithelial-mesenchymal plasticity equalizes metastatic outgrowth potential between epithelial and mesenchymal tumor cells.
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