Group 2 innate lymphoid cells (ILC2s) are distributed systemically and produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Transcriptional profiling of ILC2s from different tissues, however, grouped ILC2s according to their tissue of origin, even in the setting of combined IL-25-, IL-33-receptor-, and TSLP-receptor-deficiency. Single-cell profiling confirmed a tissue-organizing transcriptome and identified ILC2 subsets expressing distinct activating receptors, including the major subset of skin ILC2s, which were activated preferentially by IL-18. Tissue ILC2 subsets were unaltered in number and expression in germ-free mice, suggesting that endogenous, tissue-derived signals drive the maturation of ILC2 subsets by controlling expression of distinct patterns of activating receptors, thus anticipating tissue-specific perturbations occurring later in life.

Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

RESEARCH ARTICLE SUMMARY INTRODUCTION: Loss-of-function mutations in one gene copy can lead to reduced amounts of protein and, consequently, human disease, a condition termed haploinsufficiency. It is currently estimated that more than 660 genes cause human disease as a result of haploinsufficiency. The delivery of extra copies of the gene by way of gene therapy is a promising therapeutic strategy to increase genedosage in such conditions.Recombinant adeno-associated virus (rAAV) provides a promising tool for delivery of transgenes in an efficient and safe way for gene therapy. However, it has some limitations, including an optimal DNA packaging constraint of 4700 base pairs and ectopic expression. RATIONALE: Increasing the expression levels of the normal gene copy by directly targeting the endogenous gene regulatory elements that control it could potentially correct haploinsufficiency. CRISPR-mediated activation (CRISPRa), whereby a nuclease-deficient Cas9 (dCas9) is used to target a transcriptional activator to the gene’s regulatory element (promoter or enhancer), could be used for this purpose. Such an approach could overcome the ectopic expression and DNA packaging limitations of rAAV. Using obesity as a model, we tested in mice whether CRISPR-mediated activation of the existing normal copy of two different genes, Sim1 or Mc4r, where loss-of-function mutations that lead to haploinsufficiency are a major cause of human obesity, can rescue their obesity phenotype. RESULTS: We first generated a transgenic CRISPRa system using dCas9 fused to a transcriptional activator, VP64, to test whether it can rescue the obesity phenotype in a Sim1 haploinsufficient mouse model. CRISPRa targeting of the Sim1 promoter or its hypothalamusspecific enhancer, which is 270 kilobases away from the gene, in Sim1 haploinsufficient mice increased the expression of the normal copy of Sim1. This up-regulation was sufficient to rescue the obesity phenotype of Sim1 heterozygous mice and led to significantly reduced food intake and body fat content in thesemice. We assessed the off-targeting effects of CRISPRa using both RNA sequencing (RNA-seq) and Cas9 chromatin immunoprecipitation sequencing (ChIPseq) analyses. We found CRISPRa targeting to be highly specific and without any overt changes in the expression of other genes. We also observed that Sim1 up-regulation occurred only in tissues where the regulatory element (promoter or enhancer) that was being targeted was active. Although promoter-CRISPRa-targeted mice up-regulated Sim1 in all the tissues where it is expressed, the enhancer-CRISPRa-targeted mice showed Sim1 up-regulation only in the hypothalamus. We then delivered CRISPRa packaged into rAAV targeting the Sim1 promoter or its hypothalamus-specific enhancer using either Streptococcus pyogenes or the shorter Staphylococcus aureus CRISPRa system. We show that postnatal injection of CRISPRa-rAAV into the hypothalamus can up-regulate Sim1 expression and rescue the obesity phenotype in Sim1 haploinsufficient mice in a long-lasti...

Pathogenic COPA variants cause a Mendelian syndrome of immune dysregulation with elevated type I interferon signaling. COPA is a subunit of coat protein complex I (COPI) that mediates Golgi to ER transport. Missense mutations of the COPA WD40 domain impair binding and sorting of proteins targeted for ER retrieval, but how this causes disease remains unknown. Given the importance of COPA in Golgi–ER transport, we speculated that type I interferon signaling in COPA syndrome involves missorting of STING. We show that a defect in COPI transport causes ligand-independent activation of STING. Furthermore, SURF4 is an adapter molecule that facilitates COPA-mediated retrieval of STING at the Golgi. Activated STING stimulates type I interferon–driven inflammation in CopaE241K/+ mice that is rescued in STING-deficient animals. Our results demonstrate that COPA maintains immune homeostasis by regulating STING transport at the Golgi. In addition, activated STING contributes to immune dysregulation in COPA syndrome and may be a new molecular target in treating the disease.

The lack of minority data, despite a collaboration of eight universities and 13 individual laboratories, highlights the urgent need for a dedicated national effort to prioritize diversity in research. Our study expands the understanding of pharmacogenetic analyses in racially/ethnically diverse populations and advances the foundation for precision medicine in at-risk and understudied minority populations.

Idiopathic scoliosis (IS) is a common pediatric musculoskeletal disease that displays a strong female bias. By performing a genome-wide association study (GWAS) of 3,102 individuals we identify significant associations with 20p11.22 SNPs for females (P=6.89×10−9) but not males (P=0.71). This association with IS is also found in independent female cohorts from the USA and Japan (overall P=2.15×10−10, OR=1.30 (rs6137473)). Unexpectedly, the 20p11.22 IS risk alleles were previously associated with protection from early-onset alopecia, another sexually dimorphic condition. The 174 kb associated locus is distal to PAX1 which encodes paired box 1, a transcription factor involved in spine development. We identify a sequence in the associated locus with enhancer activity in zebrafish somitic muscle and spinal cord, an activity that is abolished by IS-associated SNPs. We thus identify a sexually dimorphic IS susceptibility locus, and propose the first functionally-defined candidate mutations in an enhancer that may regulate expression in specific spinal cells.

Inguinal hernia repair is one of the most commonly performed operations in the world, yet little is known about the genetic mechanisms that predispose individuals to develop inguinal hernias. We perform a genome-wide association analysis of surgically confirmed inguinal hernias in 72,805 subjects (5,295 cases and 67,510 controls) and confirm top associations in an independent cohort of 92,444 subjects with self-reported hernia repair surgeries (9,701 cases and 82,743 controls). We identify four novel inguinal hernia susceptibility loci in the regions of EFEMP1, WT1, EBF2 and ADAMTS6. Moreover, we observe expression of all four genes in mouse connective tissue and network analyses show an important role for two of these genes (EFEMP1 and WT1) in connective tissue maintenance/homoeostasis. Our findings provide insight into the aetiology of hernia development and highlight genetic pathways for studies of hernia development and its treatment.

The order Chiroptera, commonly known as bats, is the only group of mammals to have evolved the capability of flight. They are estimated to have diverged from their arboreal ancestors ~51 million years ago 1 . Their adaptions for flight include substantial specialization of the forelimb, characterized by the notable extension of digits II-V, a decrease in wing bone mineralization along the proximal-distal axis, and the retention and expansion of interdigit webbing, which is controlled by a novel complex of muscles 2,3 . Bat hindlimbs are comparatively short, with free, symmetrical digits, providing an informative contrast that can be used to highlight the genetic processes involved in bat wing formation. Previous studies that examined gene expression in developing bat forelimbs and hindlimbs reported differential expression of several genes, including Tbx3, Brinp3, Meis2, the 5′ HoxD genes and components of the Shh-Fgf signaling loop, suggesting that multiple genes and processes are involved in generating these morphological innovations [4][5][6][7][8] . Gene regulatory elements are thought to be important drivers of these changes: for example, replacement of the mouse Prx1 limb enhancer with the equivalent bat sequence resulted in elongated forelimbs 9 . However, an integrated understanding of how changes in regulatory elements, various genes and signaling pathways combine to collectively shape the bat wing remains largely elusive.To characterize the genetic differences that underlie divergence in bat forelimb and hindlimb development, we used a comprehensive, genome-wide strategy. We generated a de novo whole-genome assembly for the vesper bat, M. natalensis, for which a well-characterized stage-by-stage morphological comparison between developing bat and mouse limbs is available 10 . In this species, the developing forelimb noticeably diverges from the hindlimb from developmental stages CS15 and CS16, with clear morphological differences seen at a subsequent stage, CS17 (ref. 10). This developmental window is equivalent to embryonic day (E) 12.0 to E13.5 in mouse 4,10 . M. natalensis embryos were obtained and transcriptomic (RNA-seq) data and ChIP-seq data for both an active (acetylation of histone H3 at lysine 27, H3K27ac; refs. 11,12) and a repressive (trimethylation of histone H3 at lysine 27, H3K27me3; ref. 13) mark were generated for these three developmental stages (Fig. 1). Bats are the only mammals capable of powered flight, but little is known about the genetic determinants that shape their wings. Here we generated a genome for Miniopterus natalensis and performed RNA-seq and ChIP-seq (H3K27ac and H3K27me3) analyses on its developing forelimb and hindlimb autopods at sequential embryonic stages to decipher the molecular events that underlie bat wing development. Over 7,000 genes and several long noncoding RNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb and hindlimb, and across different stages. ChIP-seq analysis identified thousands of regions that are differentiall...