Probes for INS

A central response to insufficient cerebral oxygen delivery is a profound reprograming of metabolism, which is mainly regulated by the Hypoxia Inducible Factor (HIF). Among other responses, HIF induces the expression of the atypical mitochondrial subunit NDUFA4L2. Surprisingly, NDUFA4L2 is constitutively expressed in the brain in non-hypoxic conditions. Analysis of publicly available single cell transcriptomic (scRNA-seq) data sets coupled with high-resolution multiplexed fluorescence RNA in situ hybridization (RNA F.I.S.H.) revealed that in the murine and human brain NDUFA4L2 is exclusively expressed in mural cells with the highest levels found in pericytes and declining along the arteriole-arterial smooth muscle cell axis. This pattern was mirrored by COX4I2, another atypical mitochondrial subunit. High NDUFA4L2 expression was also observed in human brain pericytes in vitro, decreasing when pericytes are muscularized and further induced by HIF stabilization in a PHD2/PHD3 dependent manner. In vivo, Vhl conditional inactivation in pericyte targeting Ng2-cre transgenic mice dramatically induced NDUFA4L2 expression. Finally NDUFA4L2 inactivation in pericytes increased oxygen consumption and therefore the degree of HIF pathway induction in hypoxia. In conclusion our work reveals that NDUFA4L2 together with COX4I2 is a key hypoxic-induced metabolic marker constitutively expressed in pericytes coupling mitochondrial oxygen consumption and cellular hypoxia response.

Kidneys are thought to express eight different connexin isoforms (i.e., Cx 26, 30, 32, 37, 40, 43, 45, and 46), which form either hemichannels or gap junctions serving to intercellular communication and functional synchronization. Proper function of connexins has already been shown to be crucial for regulation of renal hemodynamics and renin secretion, and there is also growing evidence for connexins to play a role in pathologic conditions such as renal fibrosis or diabetic nephropathy. Therefore, exact intrarenal localization of the different connexin isoforms gains particular interest. Until now intrarenal expression of connexins has mainly been examined by immunohistochemistry, which in part generated conflicting results depending on antibodies and fixation protocols used. In this work, we used fluorescent RNAscope as an alternative technical approach to localize renal connexin mRNAs in healthy mouse kidneys. Addition of RNAscope probes for cell type specific mRNAs was used to assign connexin mRNA signals to specific cell types. We hereby found Cx26 mRNA strongly expressed in proximal tubules, Cx30 mRNA was selectively detected in the urothelium, and Cx32 mRNA was found in proximal tubules and to a lesser extent also in collecting ducts. Cx37 mRNA was mainly associated with vascular endothelium, Cx40 mRNA was largely found in glomerular mesangial and less in vascular endothelial cells, Cx43 mRNA was sparsely expressed by interstitial cells of all kidney zones, and Cx45 mRNA was predominantly found in smooth muscle cell layers of both blood vessels and ureter as well as in mesangial and interstitial (fibroblastic) cells. Cx46 mRNA could not be detected. In summary our results essentially confirm previous data on connexin expression in the renal vasculature and in glomeruli. In addition, they demonstrate strong connexin gene expression in proximal tubules, and they suggest significant connexin expression in resident tubulointerstitial cells.

Activation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood.Bulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called IN flammatory M K L1 I nteracting L ong N oncoding RNA ( INKILN ). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation and human atherosclerosis and abdominal aortic aneurysm (AAA) samples. The transcriptional regulation of INKILN was determined through luciferase reporter system and chromatin immunoprecipitation assay. Both loss- and gain-of-function approaches and multiple RNA-protein and protein-protein interaction assays were utilized to uncover the role of INKILN in VSMC proinflammatory gene program and underlying mechanisms. Bacterial Artificial Chromosome (BAC) transgenic (Tg) mice were utilized to study INKLIN expression and function in ligation injury-induced neointimal formation.INKILN expression is downregulated in contractile VSMCs and induced by human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB site within its proximal promoter. INKILN activates the proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. Mechanistically, INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks ILIβ-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1, and the luciferase activity of an NF-κB reporter. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme, USP10. INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in BAC Tg mice.These findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.

Non-alcoholic fatty liver disease (NAFLD), the most prevalent liver pathology worldwide, is intimately linked with obesity and type 2 diabetes. Liver inflammation is a hallmark of NAFLD and is thought to contribute to tissue fibrosis and disease pathogenesis. Uncoupling protein 1 (UCP1) is exclusively expressed in brown and beige adipocytes, and has been extensively studied for its capacity to elevate thermogenesis and reverse obesity. Here we identify an endocrine pathway regulated by UCP1 that antagonizes liver inflammation and pathology, independent of effects on obesity. We show that, without UCP1, brown and beige fat exhibit a diminished capacity to clear succinate from the circulation. Moreover, UCP1KO mice exhibit elevated extracellular succinate in liver tissue that drives inflammation through ligation of its cognate receptor succinate receptor 1 (SUCNR1) in liver-resident stellate cell and macrophage populations. Conversely, increasing brown and beige adipocyte content in mice antagonizes SUCNR1-dependent inflammatory signalling in the liver. We show that this UCP1-succinate-SUCNR1 axis is necessary to regulate liver immune cell infiltration and pathology, and systemic glucose intolerance in an obesogenic environment. As such, the therapeutic use of brown and beige adipocytes and UCP1 extends beyond thermogenesis and may be leveraged to antagonize NAFLD and SUCNR1-dependent liver inflammation.

After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain . Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration.Rapid regeneration of the jaw joint ligament in adult zebrafishDedifferentiation of mature ligamentocytes contributes to regenerationscRNAseq reveals dynamic ECM remodeling and immune activation during regenerationRequirement of Legumain for ECM remodeling and ligament healing.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

Breast cancer is a heterogenous disease that can be classified into multiple subtypes including the most aggressive basal-like and triple-negative subtypes. Understanding the heterogeneity within the normal mammary basal epithelial cells holds the key to inform us about basal-like cancer cell differentiation dynamics as well as potential cells of origin. Although it is known that the mammary basal compartment contains small pools of stem cells that fuel normal tissue morphogenesis and regeneration, a comprehensive yet focused analysis of the transcriptional makeup of the basal cells is lacking. We used single-cell RNA-sequencing and multiplexed RNA in-situ hybridization to characterize mammary basal cell heterogeneity. We used bioinformatic and computational pipelines to characterize the molecular features as well as predict differentiation dynamics and cell-cell communications of the newly identified basal cell states. We used genetic cell labeling to map the in vivo fates of cells in one of these states. We identified four major distinct transcriptional states within the mammary basal cells that exhibit gene expression signatures suggestive of different functional activity and metabolic preference. Our in vivo labeling and ex vivo organoid culture data suggest that one of these states, marked by Egr2 expression, represents a dynamic transcriptional state that all basal cells transit through during pubertal mammary morphogenesis. Our study provides a systematic approach to understanding the molecular heterogeneity of mammary basal cells and identifies previously unknown dynamics of basal cell transcriptional states.

The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe progressive and region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being established during cranial neural crest specification, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse potential.