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In animal models, Nipbl deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is Nipbl haploinsufficiency. Previous studies in Nipbl+/- mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type and Nipbl+/- mouse embryos at gastrulation and early cardiac crescent stages. Nipbl+/- embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition, Nanog was found to be overexpressed in all germ layers, and many gene expression changes observed in Nipbl+/- embryos could be attributed to Nanog overexpression. These findings establish a link between Nipbl deficiency, Nanog overexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects in Nipbl+/- animals and Cornelia de Lange syndrome.

Mammalian organs exhibit distinct physiology, disease susceptibility, and injury responses between the sexes. In the mouse kidney, sexually dimorphic gene activity maps predominantly to proximal tubule (PT) segments. Bulk RNA sequencing (RNA-seq) data demonstrated that sex differences were established from 4 and 8 weeks after birth under gonadal control. Hormone injection studies and genetic removal of androgen and estrogen receptors demonstrated androgen receptor (AR)-mediated regulation of gene activity in PT cells as the regulatory mechanism. Interestingly, caloric restriction feminizes the male kidney. Single-nuclear multiomic analysis identified putative cis-regulatory regions and cooperating factors mediating PT responses to AR activity in the mouse kidney. In the human kidney, a limited set of genes showed conserved sex-linked regulation, whereas analysis of the mouse liver underscored organ-specific differences in the regulation of sexually dimorphic gene expression. These findings raise interesting questions on the evolution, physiological significance, disease, and metabolic linkage of sexually dimorphic gene activity.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

Neural crest (NC) cells migrate throughout vertebrate embryos to give rise to a huge variety of cell types, but when and where lineages emerge and their regulation remain unclear. We have performed single-cell RNA sequencing (RNA-seq) of cranial NC cells from the first pharyngeal arch in zebrafish over several stages during migration. Computational analysis combining pseudotime and real-time data reveals that these NC cells first adopt a transitional state, becoming specified mid-migration, with the first lineage decisions being skeletal and pigment, followed by neural and glial progenitors. In addition, by computationally integrating these data with RNA-seq data from a transgenic Wnt reporter line, we identify gene cohorts with similar temporal responses to Wnts during migration and show that one, Atp6ap2, is required for melanocyte differentiation. Together, our results show that cranial NC cell lineages arise progressively and uncover a series of spatially restricted cell interactions likely to regulate such cell-fate decisions.

During progression from carcinoma in situ to an invasive tumor, the immune system is engaged in complex sets of interactions with various tumor cells. Tumor cell plasticity alters disease trajectories via epithelial-to-mesenchymal transition (EMT). Several of the same pathways that regulate EMT are involved in tumor-immune interactions, yet little is known about the mechanisms and consequences of crosstalk between these regulatory processes. Here we introduce a multiscale evolutionary model to describe tumor-immune-EMT interactions and their impact on epithelial cancer progression from in situ to invasive disease. Through simulation of patient cohorts in silico, the model predicts that a controllable region maximizes invasion-free survival. This controllable region depends on properties of the mesenchymal tumor cell phenotype: its growth rate and its immune-evasiveness. In light of the model predictions, we analyze EMT-inflammation-associated data from The Cancer Genome Atlas, and find that association with EMT worsens invasion-free survival probabilities. This result supports the predictions of the model, and leads to the identification of genes that influence outcomes in bladder and uterine cancer, including FGF pathway members. These results suggest new means to delay disease progression, and demonstrate the importance of studying cancer-immune interactions in light of EMT.

Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised "hierarchical-lineage" model of homeostasis.

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

The use of single-cell transcriptomics has become a major approach to delineate cell subpopulations and the transitions between them. While various computational tools using different mathematical methods have been developed to infer clusters, marker genes, and cell lineage, none yet integrate these within a mathematical framework to perform multiple tasks coherently. Such coherence is critical for the inference of cell-cell communication, a major remaining challenge. Here, we present similarity matrix-based optimization for single-cell data analysis (SoptSC), in which unsupervised clustering, pseudotemporal ordering, lineage inference, and marker gene identification are inferred via a structured cell-to-cell similarity matrix. SoptSC then predicts cell-cell communication networks, enabling reconstruction of complex cell lineages that include feedback or feedforward interactions. Application of SoptSC to early embryonic development, epidermal regeneration, and hematopoiesis demonstrates robust identification of subpopulations, lineage relationships, and pseudotime, and prediction of pathway-specific cell communication patterns regulating processes of development and differentiation.

Directional migration is inherently important for epithelial tissue regeneration and repair, but how it is precisely controlled and coordinated with cell proliferation is unclear. Here, we report that Ovol2, a transcriptional repressor that inhibits epithelial-to-mesenchymal transition (EMT), plays a crucial role in adult skin epithelial regeneration and repair. Ovol2-deficient mice show compromised wound healing characterized by aberrant epidermal cell migration and proliferation, as well as delayed anagen progression characterized by defects in hair follicle matrix cell proliferation and subsequent differentiation. Epidermal keratinocytes and bulge hair follicle stem cells (Bu-HFSCs) lacking Ovol2 fail to expand in culture and display molecular alterations consistent with enhanced EMT and reduced proliferation. Live imaging of wound explants and Bu-HFSCs reveals increased migration speed but reduced directionality, and post-mitotic cell cycle arrest. Remarkably, simultaneous deletion of Zeb1 encoding an EMT-promoting factor restores directional migration to Ovol2-deficient Bu-HFSCs. Taken together, our findings highlight the important function of an Ovol2-Zeb1 EMT-regulatory circuit in controlling the directional migration of epithelial stem and progenitor cells to facilitate adult skin epithelial regeneration and repair.

The mandibular or first pharyngeal arch forms the upper and lower jaws in all gnathostomes. A gene regulatory network that defines ventral, intermediate, and dorsal domains along the dorsal-ventral (D-V) axis of the arch has emerged from studies in zebrafish and mice, but the temporal dynamics of this process remain unclear. To define cell fate trajectories in the arches we have performed quantitative gene expression analyses of D-V patterning genes in pharyngeal arch primordia in zebrafish and mice. Using NanoString technology to measure transcript numbers per cell directly we show that, in many cases, genes expressed in similar D-V domains and induced by similar signals vary dramatically in their temporal profiles. This suggests that cellular responses to D-V patterning signals are likely shaped by the baseline kinetics of target gene expression. Furthermore, similarities in the temporal dynamics of genes that occupy distinct pathways suggest novel shared modes of regulation. Incorporating these gene expression kinetics into our computational models for the mandibular arch improves the accuracy of patterning, and facilitates temporal comparisons between species. These data suggest that the magnitude and timing of target gene expression help diversify responses to patterning signals during craniofacial development.

ABSTRACT How stem cells give rise to human interfollicular epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find at least four spatially distinct stem cell populations that decorate the top and bottom of rete ridge architecture and hold transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling through co-variance of cognate ligand-receptor pairs indicate that the basal cell populations distinctly serve as critical signaling hubs that maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest the “transitional” basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed “transitional” basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

MotivationSingle-cell RNA-sequencing (scRNA-seq) offers unprecedented resolution for studying cellular decision-making processes. Robust inference of cell state transition paths and probabilities is an important yet challenging step in the analysis of these data.ResultsHere we present scEpath, an algorithm that calculates energy landscapes and probabilistic directed graphs in order to reconstruct developmental trajectories. We quantify the energy landscape using 'single-cell energy' and distance-based measures, and find that the combination of these enables robust inference of the transition probabilities and lineage relationships between cell states. We also identify marker genes and gene expression patterns associated with cell state transitions. Our approach produces pseudotemporal orderings that are-in combination-more robust and accurate than current methods, and offers higher resolution dynamics of the cell state transitions, leading to new insight into key transition events during differentiation and development. Moreover, scEpath is robust to variation in the size of the input gene set, and is broadly unsupervised, requiring few parameters to be set by the user. Applications of scEpath led to the identification of a cell-cell communication network implicated in early human embryo development, and novel transcription factors important for myoblast differentiation. scEpath allows us to identify common and specific temporal dynamics and transcriptional factor programs along branched lineages, as well as the transition probabilities that control cell fates.Availability and implementationA MATLAB package of scEpath is available at https://github.com/sqjin/scEpath.Supplementary informationSupplementary data are available at Bioinformatics online.

As our catalog of cell states expands, appropriate characterization of these states and the transitions between them is crucial. Here we discuss the roles of intermediate cell states (ICSs) in this growing collection. We begin with definitions and discuss evidence for the existence of ICSs and their relevance in various tissues. We then provide a list of possible functions for ICSs with examples. Finally, we describe means by which ICSs and their functional roles can be identified from single-cell data or predicted from models.

Chronic inflammation is a serious risk factor for cancer; however, the routes from inflammation to cancer are poorly understood. On the basis of the processes implicated by frequently mutated genes associated with inflammation and cancer in three organs (stomach, colon, and liver) extracted from the Gene Expression Omnibus, The Cancer Genome Atlas, and Gene Ontology databases, we present a multiscale model of the long-term evolutionary dynamics leading from inflammation to tumorigenesis. The model incorporates cross-talk among interactions on several scales, including responses to DNA damage, gene mutation, cell-cycle behavior, population dynamics, inflammation, and metabolism-immune balance. Model simulations revealed two stages of inflammation-induced tumorigenesis: a precancerous state and tumorigenesis. The precancerous state was mainly caused by mutations in the cell proliferation pathway; the transition from the precancerous to tumorigenic states was induced by mutations in pathways associated with apoptosis, differentiation, and metabolism-immune balance. We identified opposing effects of inflammation on tumorigenesis. Mild inflammation removed cells with DNA damage through DNA damage-induced cell death, whereas severe inflammation accelerated accumulation of mutations and hence promoted tumorigenesis. These results provide insight into the evolutionary dynamics of inflammation-induced tumorigenesis and highlight the combinatorial effects of inflammation and metabolism-immune balance. This approach establishes methods for quantifying cancer risk, for the discovery of driver pathways in inflammation-induced tumorigenesis, and has direct relevance for early detection and prevention and development of new treatment regimes. Cancer Res; 77(22); 6429-41. ©2017 AACR.