In the 150 years since Darwin, the field of evolutionary biology has left a glaring gap in understanding how animals developed their astounding variety and complexity. The standard answer has been ...that small genetic mutations accumulate over time to produce wondrous innovations such as eyes and wings. Drawing on cutting-edge research across the spectrum of modern biology, Marc Kirschner and John Gerhart demonstrate how this stock answer is woefully inadequate. Rather they offer an original solution to the longstanding puzzle of how small random genetic change can be converted into complex, useful innovations.In a new theory they call "facilitated variation," Kirschner and Gerhart elevate the individual organism from a passive target of natural selection to a central player in the 3-billion-year history of evolution. In clear, accessible language, the authors invite every reader to contemplate daring new ideas about evolution. By closing the major gap in Darwin's theory Kirschner and Gerhart also provide a timely scientific rebuttal to modern critics of evolution who champion "intelligent design."
It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between ...genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships.
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•Cells are captured and barcoded in nanolitre droplets with high capture efficiency•Each drop hosts a hydrogel carrying photocleavable combinatorially barcoded primers•mRNA of thousands of mouse embryonic stem and differentiating cells are sequenced•Single-cell heterogeneity reveals population structure and gene regulatory linkages
Capturing single cells along with a set of uniquely barcoded primers in tiny droplets enables single-cell transcriptomics of a large number of cells in a heterogeneous population. Applying this analysis to mouse embryonic stem cells reveals their population structure, gene expression relationships, and the heterogeneous onset of differentiation.
Rescuing US biomedical research from its systemic flaws Albertsa, Bruce; Kirschner, Marc W.; Tilghman, Shirley ...
Proceedings of the National Academy of Sciences - PNAS,
04/2014, Letnik:
111, Številka:
16
Journal Article
Recenzirano
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The long-held but erroneous assumption of never-ending rapid growth in biomedical science has created an unsustainable hypercompetitive system that is discouraging even the most outstanding ...prospective students from entering our profession—and making it difficult for seasoned investigators to produce their best work. This is a recipe for long-term decline, and the problems cannot be solved with simplistic approaches. Instead, it is time to confront the dangers at hand and rethink some fundamental features of the US biomedical research ecosystem.
Many sensory systems (e.g., vision and hearing) show a response that is proportional to the fold-change in the stimulus relative to the background, a feature related to Weber's Law. Recent ...experiments suggest such a fold-change detection feature in signaling systems in cells: a response that depends on the fold-change in the input signal, and not on its absolute level. It is therefore of interest to find molecular mechanisms of gene regulation that can provide such fold-change detection. Here, we demonstrate theoretically that fold-change detection can be generated by one of the most common network motifs in transcription networks, the incoherent feedforward loop (I1-FFL), in which an activator regulates both a gene and a repressor of the gene. The fold-change detection feature of the I1-FFL applies to the entire shape of the response, including its amplitude and duration, and is valid for a wide range of biochemical parameters.
Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. We find that the Wnt receptor Frizzled2 (Fzd2) ...and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3. A gene signature regulated by this pathway predicts metastasis and overall survival in patients. We have developed an antibody to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b.
Time series of single-cell transcriptome measurements can reveal dynamic features of cell differentiation pathways. From measurements of whole frog embryos spanning zygotic genome activation through ...early organogenesis, we derived a detailed catalog of cell states in vertebrate development and a map of differentiation across all lineages over time. The inferred map recapitulates most if not all developmental relationships and associates new regulators and marker genes with each cell state. We find that many embryonic cell states appear earlier than previously appreciated. We also assess conflicting models of neural crest development. Incorporating a matched time series of zebrafish development from a companion paper, we reveal conserved and divergent features of vertebrate early developmental gene expression programs.
Ubiquitin and ubiquitin-like (Ubl) protein modifications affect protein stability, activity, and localization, but we still lack broad understanding of the functions of Ubl modifications. We have ...profiled the protein targets of ubiquitin and six additional Ubls in mitosis using a functional assay that utilizes active mammalian cell extracts and protein microarrays and identified 1,500 potential substrates; 80–200 protein targets were exclusive to each Ubl. The network structure is nonrandom, with most targets mapping to a single Ubl. There are distinct molecular functions for each Ubl, suggesting divergent biological roles. Analysis of differential profiles between mitosis and G1 highlighted a previously underappreciated role for the Ubl, FAT10, in mitotic regulation. In addition to its role as a resource for Ubl modifications, our study provides a systematic approach to analyze changes in posttranslational modifications at various cellular states.
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► High-throughput system for post-translation modification profiling in mammalian cells ► A comprehensive view of seven ubiquitin family members and their targets in mitosis ► Non-random network organization highlights diverse functions of Ubl modifications ► Analysis of differential modifications reveals a role for FAT10 in mitotic regulation
A high-throughput approach for the detection of ubiquitin family modifications in mammalian cells identifies more than 1,500 putative targets, providing information on substrate interconnections between different ubiquitin-like molecules and revealing an unexpected role for the ubiquitin-like modifier FAT10 in mitotic control.
On being the right (cell) size Ginzberg, Miriam B.; Kafri, Ran; Krischner, Marc
Science,
05/2015, Letnik:
348, Številka:
6236
Journal Article
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How cells know when they are the right size
Biologists have long recognized that cells exist in a large range of sizes. Cell size is also flexible: Cells can differentiate into another cell type with ...a very different size. External factors can also influence cell size, but the consistent size of a given cell type shows that cells have mechanisms to measure their own size and adjust their growth rate or rate of cell division to maintain uniformity. Ginzberg
et al.
review recent advances in understanding how cells know when they are at the right size.
Science
, this issue
10.1126/science.1245075
BACKGROUND
How do the different cell types in our bodies maintain their distinctive and characteristic sizes? Although much is known about the signaling networks that stimulate or suppress cell growth, such as the mammalian target of rapamycin (mTOR) pathway, this central question remains: How do a common set of pathways precisely specify the appropriate size for any given cell type and physiological condition? The precision with which size is controlled is demonstrated by the uniformity in cell size typically seen in tissues. Most epithelial tissues, for example, display a striking regularity in the size and morphology of cells, whereas size heterogeneity can be a sign of neoplastic growth.
Most work on the subject of how cell size is regulated has explored the control of cell growth and proliferation by extracellular signals, such as growth factors and cytokines. However, although these signals can dictate the mean size of cells, individual cells will inevitably deviate from that mean. Variability in cell size can arise from variability in growth rate and cell-cycle length or asymmetry in cell division. These sources of variation raise the question of whether they are counteracted by cellular mechanisms that act to increase size homogeneity. Size variation can only be reduced with processes that differentially affect cells of different sizes, despite the fact that they share the same environment. This kind of control requires that individual cells measure their own size and adjust their behavior as necessary to achieve a common target size.
ADVANCES
In this Review, we present a growing body of evidence that suggests that animal cells autonomously measure and adjust their individual sizes to maintain uniformity within a population. We discuss possible mechanisms by which this can be achieved, including the size-dependent adjustment of cell-cycle length and/or growth rate, as well as the limitations of these strategies. We summarize the progress that has been made thus far in identifying the cell’s size control machinery and highlight important unanswered questions.
The presence of mechanisms ensuring precise size specification suggests that there may be an optimal cell size for a particular cell’s function. Here, we address the question of whether cells function most efficiently when at the “right” size by examining cases in which cell size was altered naturally or experimentally. Some tissues seem to easily compensate for cell-size changes, whereas in others, cells appear to perform best at their appropriate size. We highlight examples of cell types, such as pancreatic β cells and adipocytes, in which a relationship between cell size and cell function has been observed.
OUTLOOK
We conclude by discussing the gaps in our understanding of how cell size is regulated, stressing the questions that have been most neglected. Throughout this Review, we point out the experimental challenges that have hindered progress in this field and emphasize recent technological advances that may allow us to overcome these obstacles. Last, we pose the questions that we anticipate will guide this field in the upcoming years.
How is cell size specified?
(
Left
) In populations of proliferating cells, size uniformity may be ensured by linking the processes of growth and cell-cycle progression. One way this can be accomplished is by restricting progress through a particular cell-cycle stage (for example, the G
1
/S transition) to cells that have reached a specific “target” size. (
Right
) Typical sizes of various human cell types. Cells are drawn to scale: pancreatic β cells, hepatocytes, keratinocytes, fibroblasts, and adipocytes.
ILLUSTRATION CREDIT: K. SUTLIFF/
SCIENCE
Different animal cell types have distinctive and characteristic sizes. How a particular cell size is specified by differentiation programs and physiology remains one of the fundamental unknowns in cell biology. In this Review, we explore the evidence that individual cells autonomously sense and specify their own size. We discuss possible mechanisms by which size-sensing and size-specification may take place. Last, we explore the physiological implications of size control: Why is it important that particular cell types maintain a particular size? We develop these questions through examination of the current literature and pose the questions that we anticipate will guide this field in the upcoming years.
Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic ...analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes.
Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis.
To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates.
•Genome-free proteomics identifies more than 11,000 proteins in the Xenopus laevis egg•Each protein’s expression level is predicted with approximately 2-fold precision•Many blood plasma proteins are taken up from oocyte during growth in the ovary•Web tool generates proteomic reference sets from mRNA data for any organism
Wühr et al. demonstrate the feasibility of deep proteomics, without the use of a sequenced genome, using the example of the egg of the African clawed frog Xenopus laevis. The authors identify more than 11,000 proteins and can predict each protein’s expression level with approximately 2-fold precision.
In response to Wnt stimulation, β-catenin accumulates and activates target genes. Using modeling and experimental analysis, we found that the level of β-catenin is sensitive to perturbations in the ...pathway, such that cellular variation would be expected to alter the signaling outcome. One unusual parameter was robust: the fold-change in β-catenin level (post-Wnt/pre-Wnt). In Xenopus, dorsal-anterior development and target gene expression are robust to perturbations that alter the final level but leave the fold-change intact. These suggest, first, that despite cellular noise, the cell responds reliably to Wnt stimulation by maintaining a robust fold-change in β-catenin. Second, the transcriptional machinery downstream of the Wnt pathway does not simply read the β-catenin level after Wnt stimulation but computes fold-changes in β-catenin. Analogous to Weber's Law in sensory physiology, some gene transcription networks must respond to fold-changes in signals, rather than absolute levels, which may buffer stochastic, genetic, and environmental variation.
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