Synthetic organizer cells guide development via spatial and biochemical instructions.

In vitro development relies primarily on treating progenitor cells with media-borne morphogens and thus lacks native-like spatial information. Here, we engineer morphogen-secreting organizer cells programmed to self-assemble, via cell adhesion, around mouse embryonic stem (ES) cells in defined architectures. By inducing the morphogen WNT3A and its antagonist DKK1 from organizer cells, we generated diverse morphogen gradients, varying in range and steepness.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folding generates structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, the numerous finger-like protrusions that are essential for nutrient absorption. However, the molecular and mechanical mechanisms driving the initiation and morphogenesis of villi remain a matter of debate.

Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche.

Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81 PDGFRA trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81 PDGFRA mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands.

Lymphangiocrine signals are required for proper intestinal repair after cytotoxic injury.

The intestinal epithelium undergoes continuous renewal and has an exceptional capacity to regenerate after injury. Maintenance and proliferation of intestinal stem cells (ISCs) are regulated by their surrounding niche, largely through Wnt signaling. However, it remains unclear which niche cells produce signals during different injury states, and the role of endothelial cells (ECs) as a component of the ISC niche during homeostasis and after injury has been underappreciated.

High-level correction of the sickle mutation is amplified during erythroid differentiation.

Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the β-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction.

Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one β-globin allele in more than 30% of long-term engrafting human HSCs.

Application of full-genome analysis to diagnose rare monogenic disorders.

Current genetic testenhancer and narrows the diagnostic intervals for rare diseases provide a diagnosis in only a modest proportion of cases. The Full-Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGA's utility for diagnosis of rare diseases in a clinical setting.

Towards a reference genome that captures global genetic diversity.

The current human reference genome is predominantly derived from a single individual and it does not adequately reflect human genetic diversity. Here, we analyze 338 high-quality human assemblies of genetically divergent human populations to identify missing sequences in the human reference genome with breakpoint resolution. We identify 127,727 recurrent non-reference unique insertions spanning 18,048,877 bp, some of which disrupt exons and known regulatory elements.

The Driver of Extreme Human-Specific Olduvai Repeat Expansion Remains Highly Active in the Human Genome.

Sequences encoding Olduvai protein domains (formerly DUF1220) show the greatest human lineage-specific increase in copy number of any coding region in the genome and have been associated, in a dosage-dependent manner, with brain size, cognitive aptitude, autism, and schizophrenia. Tandem intragenic duplications of a three-domain block, termed the Olduvai triplet, in four genes in the chromosomal 1q21.1-0.

Single-Cell Transcriptome Analysis of CD34 Stem Cell-Derived Myeloid Cells Infected With Human Cytomegalovirus.

Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34 hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of resistant and permissive cell types, and the cellular features characterizing the latter, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic.

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin.

Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34 hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death.

Functionally conserved enhancers with divergent sequences in distant vertebrates.

Background: To examine the contributions of sequence and function conservation in the evolution of enhancers, we systematically identified enhancers whose sequences are not conserved among distant groups of vertebrate species, but have homologous function and are likely to be derived from a common ancestral sequence. Our approach combined comparative genomics and epigenomics to identify potential enhancer sequences in the genomes of three groups of distantly related vertebrate species.

Results: We searched for sequences that were conserved within groups of closely related species but not between groups of more distant species, and were associated with an epigenetic mark of enhancer activity.

piRNA-like small RNAs mark extended 3'UTRs present in germ and somatic cells.

Background: Piwi-interacting RNAs (piRNAs) are a class of small RNAs; distinct types of piRNAs are expressed in the mammalian testis at different stages of development. The function of piRNAs expressed in the adult testis is not well established. We conducted a detailed characterization of piRNAs aligning at or near the 3' UTRs of protein-coding genes in a deep dataset of small RNAs from adult mouse testis.

Now you see it: genome methylation makes a comeback in Drosophila.

Drosophila melanogaster is often considered to lack genomic 5-methylcytosine (m(5) C), an opinion reinforced by two whole genome bisulfite-sequencing studies that failed to find m(5) C. New evidence, however, indicates that genomic methylation is indeed present in the fly, albeit in small quantities and in unusual patterns. At embryonic stage 5, m(5) C occurs in short strand-specific regions that cover ∼1% of the genome, at tissue levels suggesting a distribution restricted to a subset of nuclei.

Genome methylation in D. melanogaster is found at specific short motifs and is independent of DNMT2 activity.

Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: Its location and function have not been established. We have used a novel and highly sensitive genomewide cytosine methylation assay to detect and map genome methylation in stage 5 Drosophila embryos. The methylation we observe with this method is highly localized and strand asymmetrical, limited to regions covering ∼1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine.

Deficiency of MIWI2 (Piwil4) induces mouse erythroleukemia cell differentiation, but has no effect on hematopoiesis in vivo.

Piwi proteins and their small non-coding RNA partners are involved in the maintenance of stem cell character and genome integrity in the male germ cells of mammals. MIWI2, one of the mouse Piwi-like proteins, is expressed in the prepachytene phase of spermatogenesis during the period of de novo methylation. Absence of this protein leads to meiotic defects and a progressive loss of germ cells.

Statin-induced changes in gene expression in EBV-transformed and native B-cells.

Human lymphoblastoid cell lines (LCLs), generated through Epstein-Barr Virus (EBV) transformation of B-lymphocytes (B-cells), are a commonly used model system for identifying genetic influences on human diseases and on drug responses. We have previously used LCLs to examine the cellular effects of genetic variants that modulate the efficacy of statins, the most prescribed class of cholesterol-lowering drugs used for the prevention and treatment of cardiovascular disease. However, statin-induced gene expression differences observed in LCLs may be influenced by their transformation, and thus differ from those observed in native B-cells.

5'-YRNA fragments derived by processing of transcripts from specific YRNA genes and pseudogenes are abundant in human serum and plasma.

Small noncoding RNAs carry out a variety of functions in eukaryotic cells, and in multiple species they can travel between cells, thus serving as signaling molecules. In mammals multiple small RNAs have been found to circulate in the blood, although in most cases the targets of these RNAs, and even their functions, are not well understood. YRNAs are small (84-112 nt) RNAs with poorly characterized functions, best known because they make up part of the Ro ribonucleoprotein autoantigens in connective tissue diseases.