METHODS FOR PROFILING MOLECULES WITH AN OBJECTIVE FUNCTION

Methods relating to profiling and/or identifying molecules in a sample, particularly chemical or biological molecules contained in an experimental sample using measured data about molecules actually present and known information about candidate molecules that may be present. Information tags can be assigned to candidates. This may be achieved with a high degree of accuracy and a low false positive rate by minimising the effect of one or more possible sources of error. An objective goal (assignment) may be optimised by linear programming or by mixed integer programmin

Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells.

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.All authors were supported by EU FP7 grant DDPDGENES. S.L. was supported by European Research Council grant 261063 (BRAINCELL), Knut and Alice Wallenberg Foundation grant 2015.0041, Swedish Research Council (STARGET), and the Swedish Foundation for Strategic Research (RIF14-0057). A.Z. was supported by the Human Frontier Science Program. E.A. was supported by Swedish Research Council (VR projects: 2011-3116 and 2011-3318), Swedish Foundation for Strategic Research (SRL program), and Karolinska Institutet (SFO Thematic Center in Stem cells and Regenerative Medicine). E.A. and R.A.B. were supported by the EU FP7 grant NeuroStemcellRepair. R.A.B. was also supported by an NIHR Biomedical Research Centre award to the University of Cambridge/Addenbrookes Hospital. iCell dopaminergic neurons were a generous gift from Cellular Dynamics International. Single-cell RNA-seq servic0es were provided by the Eukaryotic Single-cell Genomics facility and the National Genomics Infrastructure at Science for Life Laboratory.This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.cell.2016.09.02

Mechanisms of cell type-specific gene transcription : Studies on the choline acetyltransferase gene

Choline acetyltransferase (ChAT) is the enzyme synthesizing the neurotransmitter acetylcholine. Previous studies from our laboratory on the regulation of the cholinergic phenotype have suggested the presence of regulatory elements in the ChAT gene controlling accurate time- and cell-specific expression. It has been shown that a 3862 base pair (bp) long, but not a 1520 bp-long, 5´ flanking segment from the rat ChAT gene upstream promoter directs cholinergic cell-specific expression of a reporter gene in cell culture, indicating that the intervening 2336 bp-long segment contains crucial regulatory elements. This upstream sequence was used to control the expression of a reporter gene in transgenic mice. Transgene expression targeted to several cholinergic regions of the central nervous system. The pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2336 bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in neurons. With the aim of increasing the expression levels and also making gene transcription easily regulatable in an on-off fashion, the tetracyclin responsive promoter system was adapted for cholinergic cell-specific expression. Directing the expression of a tetracyclin-dependent transcriptional activator (rtTA) to cholinergic neurons allowed for the dose-dependent activation of a responsive promoter by tetracyclin analogues in these cells, but not in other cell types. The 2336 bp regulatory region was analyzed for the presence of regulatory elements that direct cholinergic cell-specific gene expression. A neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region, recognized by the neuron-restrictive silencer factor (NRSF). The ChAT NRSE was inactive in neuronal cells but repressed expression in non-neuronal cells. The distal part of the ChAT gene promoter showed cholinergic-cell-specific enhancing activity, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. We observed that the reported ChAT gene expression in spermatozoa is not driven by any of the known ChAT promoters, and isolated several testis-specific cDNAs from the ChAT gene. These cDNAs code for proteins with truncated N termini. When produced in cell- free extracts and in COS cells, the proteins do not show ChAT activity and do not compete with neuronal ChAT for either substrate in competition experiments. We suggest that a novel product of unknown function lacking ChAT activity is made in testis from the gene sequence that overlaps with the ChAT gene expressed in brain. Two further studies deal with gene regulation in the testis. Androgen binding protein (ABP) mRNA levels were found to be regulated through the cycle of spermatogenesis, with a maximum during stages VIII-XI. ABP mRNA expression was also elevated upon in vivo nerve growth factor (NGF) infusion of testes, possibly through a prolongation of stages VII-VIII induced by NGF. mRNAs for regulatory subunits of protein kinase A were also strictly and individually regulated with the stages of spermatogenesis, pointing to discrete functions for the different subunits

Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq

Our understanding of the development and maintenance of tissues has been greatly aided by large-scale gene expression analysis. However, tissues are invariably complex, and expression analysis of a tissue confounds the true expression patterns of its constituent cell types. Here we describe a novel strategy to access such complex samples. Single-cell RNA-seq expression profiles were generated, and clustered to form a two-dimensional cell map onto which expression data were projected. The resulting cell map integrates three levels of organization: the whole population of cells, the functionally distinct subpopulations it contains, and the single cells themselves—all without need for known markers to classify cell types. The feasibility of the strategy was demonstrated by analyzing the transcriptomes of 85 single cells of two distinct types. We believe this strategy will enable the unbiased discovery and analysis of naturally occurring cell types during development, adult physiology, and disease

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

<div><p>Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells.</p> </div

| Optimal TSO sequences for template switching.

<p>The center panel shows the template-switching process. The template-switching event occurs in the middle of the grey circle. The investigated positions of the TSO are numbered from the template-switching site. The first three positions correspond to ribo bases and are shown in grey. The other analyzed positions are DNA bases and are depicted in black. In (a)) nucleotide preferences for all positions for RPLP1 in the RNA10N3 sample are shown. Corresponding graphs for the other transcripts and RNA spikes are shown in Figures <b>S3</b> and S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>. (b)shows the distribution of the number of guanidines seen in the sequencing output for RPLP1 in the three performed reactions. Corresponding graphs for the other transcripts and RNA spike molecules are shown in Figure <b>S5</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>. The numbers of reads for the analyzed RNA spikes and transcripts are shown in Table <b>S6</b> and Table <b>S7</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>, respectively.</p

| Optimal conditions for template switching.

<p>The qPCR-derived Ct values for the investigated conditions are shown. Please note that the y-axis scales are broken and that the graphs have different Ct value scales. Also, no error bars are shown for the no template controls (NTC) as these reactions were performed in only one tube. In contrast, the actual experiments were performed in triplicate with the standard deviation error bars displayed in the graphs. The central part of the figure illustrates the template-switching process in the setting of our STRT method. (a) The optimal TSO concentration was 1 μM and (b) shorter TSOs showed a tendency for better performance (see also Table <b>S2</b> and Figure <b>S1</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>). The green datapoint in the TSO length graph represents the published version of the TSO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#B10" target="_blank">10</a>] that is 40 bases in length. (c) shows analysis of different RT enzymes. SSII was a better choice than SSIII and, a cycled SSIII protocol (8 cycles of 50°C for 5 min and 60°C for 1 min; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#B19" target="_blank">19</a>]) did not work, possibly due to the elevated temperature periods inactivating the enzyme (see also Table <b>S2</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>). (b) The optimal SSII amount was 10 units per 10 μl reaction.</p

Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity

The murine epidermis with its hair follicles represents an invaluable model system for tissue regeneration and stem cell research. Here we used single-cell RNA-sequencing to reveal how cellular heterogeneity of murine telogen epidermis is tuned at the transcriptional level. Unbiased clustering of 1,422 single-cell transcriptomes revealed 25 distinct populations of interfollicular and follicular epidermal cells. Our data allowed the reconstruction of gene expression programs during epidermal differentiation and along the proximal-distal axis of the hair follicle at unprecedented resolution. Moreover, transcriptional heterogeneity of the epidermis can essentially be explained along these two axes, and we show that heterogeneity in stem cell compartments generally reflects this model: stem cell populations are segregated by spatial signatures but share a common basal-epidermal gene module. This study provides an unbiased and systematic view of transcriptional organization of adult epidermis and highlights how cellular heterogeneity can be orchestrated in vivo to assure tissue homeostasis