Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type–specific RNA splicing was discovered and analyzed across tissues within an individual.

Highlights d High microtubule-destabilizing activity makes mouse centromeres selfish in meiosis d Amplified BUB1 signaling enriches destabilizing activity on selfish centromeres d Selfish centromeres can modulate the BUB1 pathway by different mechanisms d Rapid progression through meiosis I can suppress centromere drive

Genetic elements compete for transmission through meiosis, when haploid gametes are created from a diploid parent. Selfish elements can enhance their transmission through a process known as meiotic drive. In female meiosis, selfish elements drive by preferentially attaching to the egg side of the spindle. This implies some asymmetry between the two sides of the spindle, but molecular mechanisms underlying spindle asymmetry are unknown. Here we found that CDC42 signaling from the cell cortex regulated microtubule tyrosination to induce spindle asymmetry. NonMendelian segregation depended on this asymmetry. Cortical CDC42 depends on polarization directed by chromosomes, which are positioned near the cortex to allow the asymmetric cell division. Thus, selfish meiotic drivers exploit the asymmetry inherent in female meiosis to bias their transmission.Genetic conflict is inherent in any haploid-diploid life cycle because genetic elements compete for transmission through meiosis. Mendel's Law of Segregation states that alleles of a gene are transmitted with equal probability, but this law can be violated by selfish genetic elements through meiotic drive, for example by eliminating competing gametes (e.g., sperm killing or spore killing) or by exploiting the asymmetry in female meiosis to increase transmission to the egg. Despite the impact of meiotic drive on evolution and genetics (1-4), the underlying mechanisms are largely unknown. Female meiosis provides a clear opportunity for selfish elements to cheat because only chromosomes that segregate to the egg can be transmitted to offspring, while the rest are degraded in polar bodies. Conceptually, female meiotic drive depends on three conditions: asymmetry in cell fate, a functional difference between homologous chromosomes that influences their segregation, and asymmetry within the meiotic spindle (5). The asymmetry in cell fate is well established (6), and chromosomal rearrangements and amplifications of repetitive sequences (e.g., centromeres) are associated with biased segregation (7-10). Asymmetry within the meiotic spindle was noted in grasshopper in 1976 (11) but not studied further.Oocyte spindles are positioned close to the cortex and oriented perpendicular to the cortex so that cytokinesis produces a large egg and a small polar body. A selfish element drives by preferentially attaching to the egg side of the spindle, implying some difference in microtubules (MTs) between the egg and cortical sides. To determine how such spindle asymmetry is regulated, using mouse oocytes as a model for meiotic drive (10, 12), we tested for asymmetry in post-translational modifications that functionally diversify MTs ( To distinguish between these possibilities, we manipulated spindle position by treating oocytes with cytochalasin B (CCB) before maturation to inhibit actin polymerization. The nucleus drifted to the cortex in 24% of these oocytes, with the spindle positioned near the cortex by 3 h after germinal vesicle breakdown (GVBD) vs. migration at 6 h under norma...

Genetic elements compete for transmission through meiosis, when haploid gametes are created from a diploid parent. Selfish elements can enhance their transmission through meiotic drive, in violation of Mendel's Law of Segregation. In female meiosis, selfish elements drive by preferentially attaching to the egg side of the spindle, which implies some asymmetry between the two sides of the spindle, but molecular mechanisms underlying spindle asymmetry are unknown. Here we show that CDC42 signaling from the cell cortex regulates microtubule tyrosination to induce spindle asymmetry, and non-Mendelian segregation depends on this asymmetry. These signals depend on cortical polarization directed by chromosomes, which are positioned near the cortex to allow the asymmetric cell division. Thus, selfish meiotic drivers exploit the asymmetry inherent in female meiosis to bias their transmission.Genetic conflict is inherent in any haploid-diploid life cycle because genetic elements compete for transmission to the offspring through meiosis, the process by which haploids are generated. Mendel's Law of Segregation states that alleles of a gene are transmitted with equal probability, but it is increasingly clear that this law is often violated, and segregation can be manipulated by selfish genetic elements through meiotic drive. Drive can occur by eliminating competing gametes that do not contain the selfish element (e.g., sperm killing or spore killing) or by exploiting the asymmetry in female meiosis to increase the transmission of the selfish element to the egg. Although the impact of meiotic drive on many aspects of evolution and genetics is now recognized, with examples widespread across eukaryotes (1-4), the underlying mechanisms are largely unknown.Female meiosis provides a clear opportunity for selfish elements to cheat because of its inherent asymmetry: only chromosomes that segregate to the egg can be transmitted to offspring, while the rest are degraded in polar bodies. Conceptually, female meiotic drive depends on three conditions: asymmetry in cell fate, a functional not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/180869 doi: bioRxiv preprint first posted online Aug. 25, 2017; 2 difference between homologous chromosomes that influences their segregation, and asymmetry within the meiotic spindle (5). The asymmetry in cell fate is well established (6), and chromosomal rearrangements and amplifications of repetitive sequences (e.g., centromeres) are associated with biased segregation (7-10). Asymmetry within the meiotic spindle was noted in grasshopper in 1976 (11), but not studied further, and molecular mechanisms regulating such asymmetry are unknown.Oocyte spindles are positioned close to the cortex and oriented perpendicular to the cortex in order to achieve the highly asymmetric cell division, so that cytokinesis produces a large egg and a small polar body (Fig. 1A). A selfish element ...

Vascular endothelial cells form the inner layer of blood vessels where they have a key role in the development and maintenance of the functional circulatory system and provide paracrine support to surrounding non-vascular cells. Technical advances in the past 5 years in single-cell genomics and in in vivo genetic labelling have facilitated greater insights into endothelial cell development, plasticity and heterogeneity. These advances have also contributed to a new understanding of the timing of endothelial cell subtype differentiation and its relationship to the cell cycle. Identification of novel tissue-specific gene expression patterns in endothelial cells has led to the discovery of crucial signalling pathways and new interactions with other cell types that have key roles in both tissue maintenance and disease pathology. In this Review, we describe the latest findings in vascular endothelial cell development and diversity, which are often supported by large-scale, single-cell studies, and discuss the implications of these findings for vascular medicine. In addition, we highlight how techniques such as single-cell multimodal omics, which have become increasingly sophisticated over the past 2 years, are being utilized to study normal vascular physiology as well as functional perturbations in disease.

Rationale: Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no methods to stimulate artery growth or regeneration in diseased hearts. Studying how arteries are built during development could illuminate strategies for re-building these vessels during ischemic heart disease. We previously found that Dach1 deletion in mouse embryos resulted in small coronary arteries. However, it was not known whether Dach1 gain-of-function would be sufficient to increase arterial vessels and whether this could benefit injury responses. Objective: We investigated how Dach1 overexpression in endothelial cells affected transcription and artery differentiation, and how it influenced recovery from myocardial infarction (MI). Methods and Results: Dach1 was genetically overexpressed in coronary endothelial cells (ECs) in either developing or adult hearts using ApjCreER. This increased the length and number of arterial end branches expanded arteries during development, in both the heart and retina, by inducing capillary ECs to differentiate and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1. In adults, Dach1 overexpression did not cause a statistically significant change artery structure prior to injury, but increased the number of perfused arteries in the injury zone post-MI. Conclusions: Our data demonstrate that increasing Dach1 is a novel method for driving artery specification and extending arterial branches, which could be explored as a means of mitigating the effects of CAD.

Asymmetric division in female meiosis creates selective pressure favoring selfish centromeres that bias their transmission to the egg. This centromere drive can explain the paradoxical rapid evolution of both centromere DNA and centromere-binding proteins despite conserved centromere function. Here, we define a molecular pathway linking expanded centromeres to histone phosphorylation and recruitment of microtubule destabilizing factors in an intraspecific hybrid, leading to detachment of selfish centromeres from spindle microtubules that would direct them to the polar body. We also introduce a second hybrid model, exploiting centromere divergence between species, and show that winning centromeres in one hybrid become losers in the other. Our results indicate that increasing destabilizing activity is a general strategy for drive, but centromeres have evolved distinct strategies to increase that activity. Furthermore, we show that drive depends on slowing meiotic progression, suggesting that a weakened meiotic spindle checkpoint evolved as a mechanism to suppress selfish centromeres.

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