Probes for INS

The murine epidermis with its hair follicles represents an invaluable model system for tissue regeneration and stem cell research. Here we used single-cell RNA-sequencing to reveal how cellular heterogeneity of murine telogen epidermis is tuned at the transcriptional level. Unbiased clustering of 1,422 single-cell transcriptomes revealed 25 distinct populations of interfollicular and follicular epidermal cells. Our data allowed the reconstruction of gene expression programs during epidermal differentiation and along the proximal-distal axis of the hair follicle at unprecedented resolution. Moreover, transcriptional heterogeneity of the epidermis can essentially be explained along these two axes, and we show that heterogeneity in stem cell compartments generally reflects this model: stem cell populations are segregated by spatial signatures but share a common basal-epidermal gene module. This study provides an unbiased and systematic view of transcriptional organization of adult epidermis and highlights how cellular heterogeneity can be orchestrated in vivo to assure tissue homeostasis.

The contributions of the innate immune system to the development of pancreatic cancer are still ill defined. Inflammatory macrophages can initiate metaplasia of pancreatic acinar cells to a duct-like phenotype (acinar-to-ductal metaplasia [ADM]), which then gives rise to pancreatic intraepithelial neoplasia (PanIN) when oncogenic KRas is present. However, it remains unclear when and how this inflammatory macrophage population is replaced by tumor-promoting macrophages. Here, we demonstrate the presence of interleukin-13 (IL-13), which can convert inflammatory into Ym1+ alternatively activated macrophages, at ADM/PanIN lesions. We further show that Ym1+ macrophages release factors, such as IL-1ra and CCL2, to drive pancreatic fibrogenesis and tumorigenesis. Treatment of mice expressing oncogenic KRas under an acinar cell-specific promoter with a neutralizing antibody for IL-13 significantly decreased the accumulation of alternatively activated macrophages at these lesions, resulting in decreased fibrosis and lesion growth.

Understanding the roles of splicing factors and splicing events during tumorigenesis would open new avenues for targeted therapies. Here we identify an oncofetal splicing factor, MBNL3, which promotes tumorigenesis and indicates poor prognosis of hepatocellular carcinoma patients. MBNL3 knockdown almost completely abolishes hepatocellular carcinoma tumorigenesis. Transcriptomic analysis revealed that MBNL3 induces lncRNA-PXN-AS1 exon 4 inclusion. The transcript lacking exon 4 binds to coding sequences of PXN mRNA, causes dissociation of translation elongation factors from PXN mRNA, and thereby inhibits PXN mRNA translation. In contrast, the transcript containing exon 4 preferentially binds to the 3' untranslated region of PXN mRNA, protects PXN mRNA from microRNA-24-AGO2 complex-induced degradation, and thereby increases PXN expression. Through inducing exon 4 inclusion, MBNL3 upregulates PXN, which mediates the pro-tumorigenic roles of MBNL3. Collectively, these data demonstrate detailed mechanistic links between an oncofetal splicing factor, a splicing event and tumorigenesis, and establish splicing factors and splicing events as potential therapeutic targets.

The somatosensory system provides animals with the ability to detect, distinguish, and respond to diverse thermal, mechanical, and irritating stimuli. While there has been progress in defining classes of neurons underlying temperature sensation and gentle touch, less is known about the neurons specific for mechanical pain. Here, we use in vivo functional imaging to identify a class of cutaneous sensory neurons that are selectively activated by high-threshold mechanical stimulation (HTMRs). We show that their optogenetic excitation evokes rapid protective and avoidance behaviors. Unlike other nociceptors, these HTMRs are fast-conducting Aδ-fibers with highly specialized circumferential endings wrapping the base of individual hair follicles. Notably, we find that Aδ-HTMRs innervate unique but overlapping fields and can be activated by stimuli as precise as the pulling of a single hair. Together, the distinctive features of this class of Aδ-HTMRs appear optimized for accurate and rapid localization of mechanical pain.

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis.

Despite decades of research on neurobiological mechanisms of psychostimulant addiction, the only effective treatment for many addicts is contingency management, a behavioral treatment that uses alternative non-drug reward to maintain abstinence. However, when contingency management is discontinued, most addicts relapse to drug use. The brain mechanisms underlying relapse after cessation of contingency management are largely unknown, and, until recently, an animal model of this human condition did not exist. Here we used a novel rat model, in which the availability of a mutually exclusive palatable food maintains prolonged voluntary abstinence from intravenous methamphetamine self-administration, to demonstrate that the activation of monosynaptic glutamatergic projections from anterior insular cortex to central amygdala is critical to relapse after the cessation of contingency management. We identified the anterior insular cortex-to-central amygdala projection as a new addiction- and motivation-related projection and a potential target for relapse prevention.

Maintaining cell fate relies on robust mechanisms that prevent the differentiation of specified cells into other cell types. This is especially critical during embryogenesis, when extensive cell proliferation, patterning, and migration events take place. Here we show that vertebrate primordial germ cells (PGCs) are protected from reprogramming into other cell types by the RNA-binding protein Dead end (Dnd). PGCs knocked down for Dnd lose their characteristic morphology and adopt various somatic cell fates. Concomitantly, they gain a gene expression profile reflecting differentiation into cells of different germ layers, in a process that we could direct by expression of specific cell-fate determinants. Importantly, we visualized these events within live zebrafish embryos, which provide temporal information regarding cell reprogramming. Our results shed light on the mechanisms controlling germ cell fate maintenance and are relevant for the formation of teratoma, a tumor class composed of cells from more than one germ layer.

Abnormal feeding often co-exists with compulsive behaviors, but the underlying neural basis remains unknown. Excessive self-grooming in rodents is associated with compulsivity. Here, we show that optogenetically manipulating the activity of lateral hypothalamus (LH) projections targeting the paraventricular hypothalamus (PVH) differentially promotes either feeding or repetitive self-grooming. Whereas selective activation of GABAergic LH→PVH inputs induces feeding, activation of glutamatergic inputs promotes self-grooming. Strikingly, targeted stimulation of GABAergic LH→PVH leads to rapid and reversible transitions to feeding from induced intense self-grooming, while activating glutamatergic LH→PVH or PVH neurons causes rapid and reversible transitions to self-grooming from voracious feeding induced by fasting. Further, specific inhibition of either LH→PVH GABAergic action or PVH neurons reduces self-grooming induced by stress. Thus, we have uncovered a parallel LH→PVH projection circuit for antagonistic control of feeding and self-grooming through dynamic modulation of PVH neuron activity, revealing a common neural pathway that underlies feeding and compulsive behaviors.

Mouse heart development arises from Mesp1 expressing cardiovascular progenitors (CPs) that are specified during gastrulation. The molecular processes that control early regional and lineage segregation of CPs have been unclear. Here, we performed single cell RNA-sequencing of WT and Mesp1 null CPs in mice. We showed that populations of Mesp1 CPs are molecularly distinct and span the continuum between epiblast and later mesodermal cells including hematopoietic progenitors. Single cell transcriptome analysis of Mesp1-deficient CPs showed that Mesp1 is required for the exit from the pluripotent state and the induction of the cardiovascular gene expression program. We identified distinct populations of Mesp1 CPs that correspond to progenitors committed to different cell lineages and regions of the heart, identifying the molecular features associated with early lineage restriction and regional segregation of the heart at the early stage of mouse gastrulation.

Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat AT1 cells, which mediate gas exchange, and AT2 cells, which secret surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: maintaining Wnt signaling prevents transdifferentiation whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting 'bulk' AT2s as progenitors. Thus, individual AT2 stem cells reside in single cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface, and is coopted in cancer.

Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation.