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Background Human pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states. Results Here, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T) + mesendoderm toward a CXCR4 + DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation. Conclusions We report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems.

The ability to quantify cellular heterogeneity is a major advantage of single-cell technologies. However, statistical methods often treat cellular heterogeneity as a nuisance. We present a novel method to characterize differences in expression in the presence of distinct expression states within and among biological conditions. We demonstrate that this framework can detect differential expression patterns under a wide range of settings. Compared to existing approaches, this method has higher power to detect subtle differences in gene expression distributions that are more complex than a mean shift, and can characterize those differences. The freely available R package scDD implements the approach.

SCnorm normalizes single-cell RNA-seq data for improved downstream analyses such as differential expression and cell-state discrimination. The normalization of RNA-seq data is essential for accurate downstream inference, but the assumptions upon which most normalization methods are based are not applicable in the single-cell setting. Consequently, applying existing normalization methods to single-cell RNA-seq data introduces artifacts that bias downstream analyses. To address this, we introduce SCnorm for accurate and efficient normalization of single-cell RNA-seq data.

Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis , we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5′-NNNNGATT-3′ protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.

Background High-throughput expression profiling experiments with ordered conditions (e.g. time-course or spatial-course) are becoming more common for studying detailed differentiation processes or spatial patterns. Identifying dynamic changes at both the individual gene and whole transcriptome level can provide important insights about genes, pathways, and critical time points. Results We present an R package, Trendy, which utilizes segmented regression models to simultaneously characterize each gene’s expression pattern and summarize overall dynamic activity in ordered condition experiments. For each gene, Trendy finds the optimal segmented regression model and provides the location and direction of dynamic changes in expression. We demonstrate the utility of Trendy to provide biologically relevant results on both microarray and RNA-sequencing (RNA-seq) datasets. Conclusions Trendy is a flexible R package which characterizes gene-specific expression patterns and summarizes changes of global dynamics over ordered conditions. Trendy is freely available on Bioconductor with a full vignette at https://bioconductor.org/packages/release/bioc/html/Trendy.html .

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Abstract The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

Somitogenesis is a hallmark feature of all vertebrates and some invertebrate species that involves the periodic formation of block-like structures called somites. Somites are transient embryonic segments that eventually establish the entire vertebral column. A highly conserved molecular oscillator called the segmentation clock underlies this periodic event and the pace of this clock regulates the pace of somite formation. Although conserved signaling pathways govern the clock in most vertebrates, the mechanisms underlying the species-specific divergence in various clock characteristics remain elusive. For example, the segmentation clock in classical model species such as zebrafish, chick, and mouse embryos tick with a periodicity of ∼30, ∼90, and ∼120 min respectively. This enables them to form the species-specific number of vertebrae during their overall timespan of somitogenesis. Here, we perform a systematic review of the species-specific features of the segmentation clock with a keen focus on mouse embryos. We perform this review using three different perspectives: Notch-responsive clock genes, ligand-receptor dynamics, and synchronization between neighboring oscillators. We further review reports that use non-classical model organisms and in vitro model systems that complement our current understanding of the segmentation clock. Our review highlights the importance of comparative developmental biology to further our understanding of this essential developmental process.

The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases. Older people have an increased risk of developing many diseases, such as diabetes and age-related macular degeneration (which is often shortened to AMD). This suggests that changes that occur during normal aging may some how be linked to how such diseases develop. However, the molecular mechanisms responsible for these links are not clear. AMD causes damage to the retina of the eye, which can lead to visual loss in older people. To investigate the link between aging and age-dependent diseases, Lee et al. used mutant mice whose retina of the eye ages more quickly than normal mice and are prone to developing an eye condition that is similar to AMD. The experiments show that these mice have a mutation in a gene called Tmem135 that is responsible for these visual problems. Tmem135 regulates the size of cell compartments called mitochondria, which produce energy for the cell. This affects the ability of the mitochondria to work properly and makes the cells more sensitive to environmental stress, which in turn makes the retina age more quickly. The findings of Lee et al. show that Tmem135 is a critical link between aging and an AMD-like condition in mice. Furthermore, the experiments suggest that defects in mitochondria may accelerate the normal pace of aging and lead to AMD and other age-dependent diseases. Further studies are needed to find out exactly what role Tmem135 plays in mitochondria and whether it also contributes to the aging of other parts of the body.