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In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear.sup.1,2. Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction.sup.3-7, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGF[beta]-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGF[beta]-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease. BET proteins regulate a reversible transcriptional switch that governs fibroblast activation in heart disease through the transcription factor MEOX1., Author(s): Michael Alexanian [sup.1] , Pawel F. Przytycki [sup.1] , Rudi Micheletti [sup.2] , Arun Padmanabhan [sup.1] [sup.3] , Lin Ye [sup.1] , Joshua G. Travers [sup.4] , Barbara Gonzalez-Teran [...]

Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells.sup.1, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors.sup.2,3 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice.sup.4. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior-posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects. Single-cell RNA-sequencing analysis reveals functions of lineage-specifying transcription factors underlying congenital defects in heart development., Author(s): T. Yvanka de Soysa [sup.1] [sup.2] [sup.3] , Sanjeev S. Ranade [sup.1] [sup.3] , Satoshi Okawa [sup.4] [sup.5] , Srikanth Ravichandran [sup.4] , Yu Huang [sup.1] [sup.3] , Hazel [...]

Diseases caused by gene haploinsufficiency in humans commonly lack a phenotype in mice that are heterozygous for the orthologous factor, impeding the study of complex phenotypes and critically limiting the discovery of therapeutics. Laboratory mice have longer telomeres relative to humans, potentially protecting against age-related disease caused by haploinsufficiency. Here, we demonstrate that telomere shortening in NOTCH1-haploinsufficient mice is sufficient to elicit age-dependent cardiovascular disease involving premature calcification of the aortic valve, a phenotype that closely mimics human disease caused by NOTCH1 haploinsufficiency. Furthermore, progressive telomere shortening correlated with severity of disease, causing cardiac valve and septal disease in the neonate that was similar to the range of valve disease observed within human families. Genes that were dysregulated due to NOTCH1 haploinsufficiency in mice with shortened telomeres were concordant with proosteoblast and proinflammatory gene network alterations in human NOTCH1 heterozygous endothelial cells. These dysregulated genes were enriched for telomere-contacting promoters, suggesting a potential mechanism for telomere-dependent regulation of homeostatic gene expression. These findings reveal a critical role for telomere length in a mouse model of age-dependent human disease and provide an in vivo model in which to test therapeutic candidates targeting the progression of aortic valve disease., Introduction Use of animal models to investigate age-dependent diseases in humans typically requires long-term aging. Furthermore, human diseases caused by genetic haploinsufficiency often fail to be modeled at any age [...]

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals (1). It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive (2). Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes (3,4). It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron (4-6); however, major aspects of touch sensation remain intact without Merkel cell activity (4,7). Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation., Dorsal root ganglion (DRG) neurons have pseudounipolar axons that terminate in the skin where they form specialized mechanoreceptors that are tuned to detect mechanical forces such as stretch, indentation and [...]

Respiratory dysfunction is a notorious cause of perinatal mortality in infants and sleep apnoea in adults, but the mechanisms of respiratory control are not clearly understood. Mechanical signals transduced by airway-innervating sensory neurons control respiration; however, the physiological significance and molecular mechanisms of these signals remain obscured. Here we show that global and sensory neuron-specific ablation of the mechanically activated ion channel Piezo2 causes respiratory distress and death in newborn mice. Optogenetic activation of Piezo2[sup.+] vagal sensory neurons causes apnoea in adult mice. Moreover, induced ablation of Piezo2 in sensory neurons of adult mice causes decreased neuronal responses to lung inflation, an impaired HeringBreuer mechanoreflex, and increased tidal volume under normal conditions. These phenotypes are reproduced in mice lacking Piezo2 in the nodose ganglion. Our data suggest that Piezo2 is an airway stretch sensor and that Piezo2-mediated mechanotransduction within various airway-innervating sensory neurons is critical for establishing efficient respiration at birth and maintaining normal breathing in adults., Author(s): Keiko Nonomura [1]; Seung-Hyun Woo [1]; Rui B. Chang [2]; Astrid Gillich [3]; Zhaozhu Qiu [1, 4]; Allain G. Francisco [1]; Sanjeev S. Ranade [1]; Stephen D. Liberles (corresponding [...]