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Neural crest cells develop into tissues ranging from craniofacial bones to peripheral neurons. Combining single-cell RNA sequencing with spatial transcriptomics, Soldatov et al. analyzed how neural crest cells in mouse embryos decide among the various fates available to them (see the Perspective by Mayor). These multipotent cells become biased toward a given fate early on and step through a progression of binary decisions as their fate is refined. Competing fate programs coexist until increased synchronization favors one and repression disfavors the other. Science , this issue p. eaas9536 ; see also p. 937 Differentiation of cells of the neural crest proceeds through binary decisions that restrict and refine developmental paths. Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor Twist1. The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs.

Tissue-resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that erythro-myeloid progenitors in mice generate premacrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day 9.5 in a chemokine-receptor–dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue-specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis, as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for patients with chronic lymphocytic leukemia (CLL), despite extensive heterogeneity in this disease. To define the underlining regulatory dynamics, we analyze high-resolution time courses of ibrutinib treatment in patients with CLL, combining immune-phenotyping, single-cell transcriptome profiling, and chromatin mapping. We identify a consistent regulatory program starting with a sharp decrease of NF-κB binding in CLL cells, which is followed by reduced activity of lineage-defining transcription factors, erosion of CLL cell identity, and acquisition of a quiescence-like gene signature. We observe patient-to-patient variation in the speed of execution of this program, which we exploit to predict patient-specific dynamics in the response to ibrutinib based on the pre-treatment patient samples. In aggregate, our study describes time-dependent cellular, molecular, and regulatory effects for therapeutic inhibition of B cell receptor signaling in CLL, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring. Ibrutinib, a Bruton tyrosine kinase inhibitor, provides effective treatment for chronic lymphocytic leukemia (CLL). Here, the authors describe time-dependent molecular changes to malignant cells and to the immune system in patients undergoing ibrutinib therapy, with can be used for therapy monitoring.

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation. In most mammals, imprinted genes contain epigenetic marks that differ in each parental genome and control their parent-of-origin-specific expression. Here, the authors map imprinted genes in mouse preimplantation embryos and find that imprinted gene expression in blastocysts is mainly dependent on Polycomb-mediated H3K27me3-associated gene silencing.

The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.

The hypothalamus is a brain region rich in functionally segregated neurons. Here Romanov and colleagues use single-cell RNA sequencing to distinguish 62 neuronal subtypes and define their neuropeptide and neurotransmitter makeup. They then show that onecut-3-containing dopamine neurons populate the periventricular area and are wired into the circadian circuitry. The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S + neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S + inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

We performed a multi-ethnic Epigenome Wide Association study on 22,774 individuals to describe the DNA methylation signature of chronic low-grade inflammation as measured by C-Reactive protein (CRP). We find 1,511 independent differentially methylated loci associated with CRP. These CpG sites show correlation structures across chromosomes, and are primarily situated in euchromatin, depleted in CpG islands. These genomic loci are predominantly situated in transcription factor binding sites and genomic enhancer regions. Mendelian randomization analysis suggests altered CpG methylation is a consequence of increased blood CRP levels. Mediation analysis reveals obesity and smoking as important underlying driving factors for changed CpG methylation. Finally, we find that an activated CpG signature significantly increases the risk for cardiometabolic diseases and COPD. Chronic inflammation, marked by C-reactive protein, has been associated with changes in methylation, but the causal relationship is unclear. Here, the authors perform a Epigenome-wide association meta-analysis for C-reactive protein levels and find that these methylation changes are likely the consequence of inflammation and could contribute to disease.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions—once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Serum concentration of hepatic enzymes are linked to liver dysfunction, metabolic and cardiovascular diseases. We perform genetic analysis on serum levels of alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) using data on 437,438 UK Biobank participants. Replication in 315,572 individuals from European descent from the Million Veteran Program, Rotterdam Study and Lifeline study confirms 517 liver enzyme SNPs. Genetic risk score analysis using the identified SNPs is strongly associated with serum activity of liver enzymes in two independent European descent studies (The Airwave Health Monitoring study and the Northern Finland Birth Cohort 1966). Gene-set enrichment analysis using the identified SNPs highlights involvement in liver development and function, lipid metabolism, insulin resistance, and vascular formation. Mendelian randomization analysis shows association of liver enzyme variants with coronary heart disease and ischemic stroke. Genetic risk score for elevated serum activity of liver enzymes is associated with higher fat percentage of body, trunk, and liver and body mass index. Our study highlights the role of molecular pathways regulated by the liver in metabolic disorders and cardiovascular disease. Plasma levels of liver enzymes provide insights into hepatic function and related diseases. Here, the authors perform a genome-wide association study on three liver enzymes, identifying genetic variants associated with their plasma concentration as well as links to metabolic and cardiovascular diseases.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates 1 , 2 . Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development 3 . Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells 4 . We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning 5 in the hypothalamus. In particular, loss of SLIT–ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life. Single-cell RNA sequencing reveals molecular determinants of the developmental programs that orchestrate the intermingling of neuronal subtypes in the hypothalamus.