Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals.

Only a very small fraction of long noncoding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but the absence of lncRNA annotations in non-model organisms has precluded comparative analyses. Here we present a large-scale evolutionary study of lncRNA repertoires and expression patterns, in 11 tetrapod species. We identify approximately 11,000 primate-specific lncRNAs and 2,500 highly conserved lncRNAs, including approximately 400 genes that are likely to have originated more than 300 million years ago. We find that lncRNAs, in particular ancient ones, are in general actively regulated and may function predominantly in embryonic development. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network suggests potential functions for lncRNAs in fundamental processes such as spermatogenesis and synaptic transmission, but also in more specific mechanisms such as placenta development through microRNA production.

Colonization by Lactobacillus in the female genital tract is thought to be critical for maintaining genital health. However, little is known about how genital microbiota influence host immune function and modulate disease susceptibility. We studied a cohort of asymptomatic young South African women and found that the majority of participants had genital communities with low Lactobacillus abundance and high ecological diversity. High diversity communities strongly correlated with genital pro-inflammatory cytokine concentrations in both cross-sectional and longitudinal analyses. Transcriptional profiling suggested that genital antigen presenting cells sense gram-negative bacterial products in situ via Toll-like receptor 4 signaling, contributing to genital inflammation through activation of the NF-κB signaling pathway and recruitment of lymphocytes by chemokine production. Our study proposes a mechanism by which cervicovaginal microbiota impact genital inflammation and thereby may affect a woman's reproductive health, including her risk of acquiring HIV.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression, yet their origins and functional evolution in mammals remain little understood due to the lack of appropriate comparative data. Using RNA sequencing, we have generated extensive and comparable miRNA data for five organs in six species that represent all main mammalian lineages and birds (the evolutionary outgroup) with the aim to unravel the evolution of mammalian miRNAs. Our analyses reveal an overall expansion of miRNA repertoires in mammals, with threefold accelerated birth rates of miRNA families in placentals and marsupials, facilitated by the de novo emergence of miRNAs in host gene introns. Generally, our analyses suggest a high rate of miRNA family turnover in mammals with many newly emerged miRNA families being lost soon after their formation. Selectively preserved mammalian miRNA families gradually evolved higher expression levels, as well as altered mature sequences and target gene repertoires, and were apparently mainly recruited to exert regulatory functions in nervous tissues. However, miRNAs that originated on the X chromosome evolved high expression levels and potentially diverse functions during spermatogenesis, including meiosis, through selectively driven duplicationdivergence processes. Overall, our study thus provides detailed insights into the birth and evolution of mammalian miRNA genes and the associated selective forces.

A large-scale comparative gene expression study reveals the different ways in which the chromosome-wide gene dosage reductions resulting from sex chromosome differentiation events were compensated during mammalian and avian evolution.

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies.

Directed differentiation of cells in vitro is1 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/003236 doi: bioRxiv preprint first posted online Mar. 5, 2014; Soumillon et al. Characterization of directed differentiation by high-throughput single-cell RNA-SeqSingle-cell transcriptome profiling enabled by next-generation sequencing has recently emerged as a promising tool for characterization of heterogeneous cell populations 1 , but routine adoption will require development of protocols that allow unbiased profiling of large numbers of cells at reasonable cost. To enable efficient characterization of thousands of single cells at a time, we developed a 3' digital gene expression (3' DGE) RNA-Seq protocol that we refer to as SCRB-Seq (single cell RNA barcoding and sequencing; Supplementary Methods, Figure S1 and Table S1). Building on recent advances 2-6 , SCRB-Seq relies on a template-switching reverse transcriptase to convert poly(A)+ mRNA from isolated single cells to cDNA decorated with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs) 7 . Decorated cDNA from multiple cells are then pooled, amplified and prepared for multiplexed sequencing using a modified transposon-based fragmentation approach that enriches for 3' ends and preserves strand information. SCRB-Seq is specifically optimized for surveying mRNA from large cell numbers using minimal reagents, reagent transfers and sequencing reads per cell, with the aim of characterizing the major patterns of gene expression variation across heterogeneous populations in a cost-efficient manner. It requires approximately two times fewer enzymatic reactions, purifications and liquid transfer steps than a previous high-throughput protocol 8 and is complementary to protocols that are optimized for deep, full-length transcriptome coverage 9 .To demonstrate SCRB-Seq, we applied it to characterize a primary human adipose-derived stem/stromal cell (hASC) 10 differentiation system (see Supplementary Materials and Figure S2). In vitro adipogenesis is both a general model of lineage commitment and an important source of cells for research on metabolic disorders 11,12 . A variety of cell populations can be induced to differentiate by cocktails of adipogenic hormones and growth factors, but their yields of lipid-filled, adipocyte-like cells are highly variable. It remains unclear to what extent this variability reflects heterogeneity in the starting populations, stochastic responses to imperfect differentiation stimuli or other factors. The great majority of adipogenesis research over the last three decades has therefore relied on the immortalized murine 3T3-L1 cell line, which supports near complete conversion to adipocyte-like cells 13 . Numerous molecular differences have, however, been found between this cell line and hASCs 14 . Single-cell profiling should help clarify the origin and relevance of these differenc...