Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral overlap between fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using fluorescence in situ hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells is a natural approach to bring systems biology into single cells.
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"It is inline with this prevailing view, therefore, that twi is consistently the most abundant mesoderm gene quantified. Using FISH to count single points of fluorescence can be challenging, with probe design and microscopy techniques affecting the counts (FEMINO et al. 1998; RAJ et al. 2008; LUBECK and CAI 2012). In addition, the combination of dense points of fluorescence signal making it difficult to distinguish individual spots, and the use of a threshold to exclude fluorescent signal, may reduce the number of transcripts counted and account for the differences between our quantification and the numbers calculated for bcd and sna. "
[Show abstract][Hide abstract]ABSTRACT: During embryonic development of Drosophila melanogaster, the Maternal to Zygotic Transition (MZT) marks a significant and rapid turning point when zygotic transcription begins and control of development is transferred from maternally deposited transcripts. Characterizing the sequential activation of the genome during the MZT requires precise timing and a sensitive assay to measure changes in expression. We utilized the NanoString nCounter instrument, which directly counts mRNA transcripts without reverse transcription or amplification, to study over 70 genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, dividing the MZT into 10 time points. Transcripts were quantified for every gene studied at all time points, providing the first data set of absolute numbers of transcripts during Drosophila development. We found that gene expression changes quickly during the MZT, with early Nuclear Cycle (NC) 14 the most dynamic time for the embryo. twist is one of the most abundant genes in the entire embryo and we use mutants to quantitatively demonstrate how it cooperates with Dorsal to activate transcription and is responsible for some of the rapid changes in transcription observed during early NC14. We also uncovered elements within the gene regulatory network that maintain precise transcript levels for sets of genes that are spatiotemporally co-transcribed within the presumptive mesoderm or dorsal ectoderm. Using this new data, we show that a fine-scale, quantitative analysis of temporal gene expression can provide new insights into developmental biology by uncovering trends in gene networks including coregulation of target genes and specific temporal input by transcription factors.
"Such approaches may be adapted to other epigenomic assays, including Hi-C, DamID, and ChIP, thus providing a way forward to achieving the required throughput to confidently define new cell types, or organize populations of cells going through some biological process into some sort of ''pseudotime. " It may also be useful to consider the marriage of single-cell biochemical techniques with complimentary microscopeacquired in situ transcriptomic datasets  . In situ transcriptomics may, for example, be necessary to properly spatially organize large populations of tissue-derived nuclei in some biologically meaningful way. "
[Show abstract][Hide abstract]ABSTRACT: The manner by which eukaryotic genomes are packaged into nuclei while maintaining crucial nuclear functions remains one of the fundamental mysteries in biology. Over the last ten years, we have witnessed rapid advances in both microscopic and nucleic acid-based approaches to map genome architecture, and the application of these approaches to the dissection of higher-order chromosomal structures has yielded much new information. It is becoming increasingly clear, for example, that interphase chromosomes form stable, multilevel hierarchical structures. Among them, self-associating domains like so-called topologically associating domains (TADs) appear to be building blocks for large-scale genomic organization. This review describes features of these broadly-defined hierarchical structures, insights into the mechanisms underlying their formation, our current understanding of how interactions in the nuclear space are linked to gene regulation, and important future directions for the field.
"Picking up old threads, Kauffman and colleagues made efforts to identify signatures of criticality in genetic networks155156157, while my colleagues and I have argued that one can see such signatures in the the statistical and dynamical behavior of the gap gene network . I think all will be clearer when we finally have tools that allow us to measure simultaneously the expression levels of many genes, in single cells, with a resolution significantly better than the intrinsic noise levels, and these are just emerging [159, 160] . nearly parameter–free explanation for the essential nonlinearities of auditory perception174175176; and more. "
[Show abstract][Hide abstract]ABSTRACT: Theoretical physics is the search for simple and universal mathematical
descriptions of the natural world. In contrast, much of modern biology is an
exploration of the complexity and diversity of life. For many, this contrast is
prima facie evidence that theory, in the sense that physicists use the word, is
impossible in a biological context. For others, this contrast serves to
highlight a grand challenge. I'm an optimist, and believe (along with many
colleagues) that the time is ripe for the emergence of a more unified
theoretical physics of biological systems, building on successes in thinking
about particular phenomena. In this essay I try to explain the reasons for my
optimism, through a combination of historical and modern examples.