Gt(ROSA)26Sor - gene trap ROSA 26, Philippe Soriano
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Publication
Journal: Molecular Metabolism
December/9/2018
Abstract
Brown adipose tissue (BAT) thermogenesis depends on the mobilization and oxidation of fatty acids from intracellular lipid droplets (LD) within brown adipocytes (BAs); however, the identity and function of LD proteins that control BAT lipolysis remain incomplete. Proteomic analysis of mouse BAT subcellular fractions identified vacuolar protein sorting 13C (VPS13C) as a novel LD protein. The aim of this work was to investigate the role of VPS13C on BA LDs.
Biochemical fractionation and high resolution confocal and immuno-transmission electron microscopy (TEM) were used to determine the subcellular distribution of VPS13C in mouse BAT, white adipose tissue, and BA cell culture. Lentivirus-delivered shRNA was used to determine the role of VPS13C in regulating lipolysis and gene expression in cultured BA cells.
We found that VPS13C is highly expressed in mouse BAT where it is targeted to multilocular LDs in a subspherical subdomain. In inguinal white adipocytes, VPS13C was mainly observed on small LDs and β3-adrenergic stimulation increased VPS13C in this depot. Silencing of VPS13C in cultured BAs decreased LD size and triglyceride content, increased basal free fatty acid release, augmented the expression of thermogenic genes, and enhanced the lipolytic potency and efficacy of isoproterenol. Mechanistically, we found that BA lipolysis required activation of adipose tissue triglyceride lipase (ATGL) and that loss of VPS13C greatly increased the association of ATGL to LDs.
VPS13C is present on BA LDs where is targeted to a distinct subdomain. VPS13C limits the access of ATGL to LD and loss of VPS13C elevates lipolysis and promotes oxidative gene expression.
Publication
Journal: Nature Communications
December/9/2018
Abstract
Excess caloric intake results in increased fat accumulation and an increase in energy expenditure via diet-induced adaptive thermogenesis; however, the underlying mechanisms controlling these processes are unclear. Here we identify the neuropeptide FF receptor-2 (NPFFR2) as a critical regulator of diet-induced thermogenesis and bone homoeostasis. Npffr2-/- mice exhibit a stronger bone phenotype and when fed a HFD display exacerbated obesity associated with a failure in activating brown adipose tissue (BAT) thermogenic response to energy excess, whereas the activation of cold-induced BAT thermogenesis is unaffected. NPFFR2 signalling is required to maintain basal arcuate nucleus NPY mRNA expression. Lack of NPFFR2 signalling leads to a decrease in BAT thermogenesis under HFD conditions with significantly lower UCP-1 and PGC-1α levels in the BAT. Together, these data demonstrate that NPFFR2 signalling promotes diet-induced thermogenesis via a novel hypothalamic NPY-dependent circuitry thereby coupling energy homoeostasis with energy partitioning to adipose and bone tissue.
Publication
Journal: Nature Cell Biology
December/8/2018
Abstract
Tissue homeostasis is sustained by stem cell self-renewal and differentiation. How stem cells coordinately differentiate into multiple cell types is largely unclear. Recent studies underline the heterogeneity among stem cells or common progenitors, suggesting that coordination occurs at the stem cell/progenitor level1-4. Here, by tracking and manipulating the same stem cells and their progeny at the single-cell level in live mice, we uncover an unanticipated flexibility of homeostatic stem cell differentiation in hair follicles. Although stem cells have been shown to be flexible upon injury, we demonstrate that hair germ stem cells at the single-cell level can flexibly establish all of the differentiation lineages even in uninjured conditions. Furthermore, stem cell-derived hair progenitors in the structure called matrix, previously thought to be unipotent, flexibly change differentiation outcomes as a consequence of unexpected dynamic relocation. Finally, the flexible cell fate determination mechanism maintains normal differentiation and tissue architecture against an ectopic differentiation stimulus induced by Wnt activation. This work provides a model of continual fate channelling and late commitment of stem cells to achieve coordinated differentiation and robust tissue architecture.
Publication
Journal: Neuron
December/7/2018
Abstract
Retinal waves are bursts of correlated activity that occur prior to eye opening and provide a critical source of activity that drives the refinement of retinofugal projections. Retinal waves are thought to be initiated spontaneously with their spatiotemporal features dictated by immature neural circuits. Here we demonstrate that, during the second postnatal week in mice, changes in light intensity dictate where and when a subset of retinal waves are triggered via activation of conventional photoreceptors. Propagation properties of triggered waves are indistinguishable from spontaneous waves, indicating that they are activating the same retinal circuits. Using whole-brain imaging techniques, we demonstrate that light deprivation prior to eye opening diminishes eye-specific segregation of the retinal projections to the dorsolateral geniculate nucleus of the thalamus, but not other retinal targets. These data indicate that light that passes through the closed eyelids plays a critical role in the development of the image-forming visual system.
Publication
Journal: American Journal of Pathology
December/7/2018
Abstract
Past studies have identified hepatic tumors with mixed hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) characteristics that have a more aggressive behavior and a poorer prognosis than classic HCC. Whether this pathologic heterogeneity is due to a cell of origin of bipotent liver progenitors or the plasticity of cellular constituents comprising these tumors remains debated. In this study, we investigated the potential acquisition of CC-like traits during advanced development of HCC in mice. Primary and rare high-grade HCC developed in a genetic mouse model. A mouse model of highly efficient HCC invasion and metastasis by orthotopic transplantation of liver cancer organoids propagated from primary tumors in the genetic model was further developed. Invasive/metastatic tumors developed in both models closely recapitulated advanced human HCC and displayed a striking acquisition of CC-related pathologic and molecular features, which was absent in the primary HCC tumors. Our study directly demonstrates the pathologic evolution of HCC during advanced tumor development, providing the first evidence that tumors with mixed HCC and CC features, or at least a subset of these tumors, represent a more advanced developmental stage of HCC. Finally, liver cancer organoid-generated high-grade tumors exhibited significantly increased extracellular vesicle secretion, suggesting that identifying tumor-specific extracellular vesicle proteins in plasma may be a promising tool for liver cancer detection.
Publication
Journal: Transgenic Research
December/6/2018
Abstract
Reporter genes play important roles in transgenic research. LacZ is a widely used reporter gene that encodes Escherichia coli β-galactosidase, an enzyme that is well known for its ability to hydrolyze X-gal into a blue product. It is unknown whether transgenic LacZ has any adverse effects. R26R reporter mice, containing a LacZ reporter gene, were generated to monitor the in vivo recombination activity of various transgenic Cre recombinase via X-gal staining. P0-Cre is expressed in neural crest-derived cells, which give rise to the majority of the craniofacial bones. Herein, we report that 12% of the R26R reporter mice harboring P0-Cre had unexpected mid-facial developmental defects manifested by the asymmetrical growth of some facial bones, thus resulting in tilted mid-facial structure, shorter skull length, and malocclusion. Histological examination showed a disorganization of the frontomaxillary suture, which may at least partly explain the morphological defect in affected transgenic mice. Our data calls for the consideration of the potential in vivo adverse effects caused by transgenic β-galactosidase.
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Publication
Journal: Nature Communications
December/6/2018
Abstract
GABAergic interneurons in the hippocampus provide for local and long-distance coordination of neurons in functionally connected areas. Vasoactive intestinal peptide-expressing (VIP+) interneurons occupy a distinct niche in circuitry as many of them specialize in innervating GABAergic cells, thus providing network disinhibition. In the CA1 hippocampus, VIP+ interneuron-selective cells target local interneurons. Here, we discover a type of VIP+ neuron whose axon innervates CA1 and also projects to the subiculum (VIP-LRPs). VIP-LRPs show specific molecular properties and target interneurons within the CA1 area but both interneurons and pyramidal cells within subiculum. They are interconnected through gap junctions but demonstrate sparse spike coupling in vitro. In awake mice, VIP-LRPs decrease their activity during theta-run epochs and are more active during quiet wakefulness but not coupled to sharp-wave ripples. Together, the data provide evidence for VIP interneuron molecular diversity and functional specialization in controlling cell ensembles along the hippocampo-subicular axis.
Publication
Journal: eNeuro
December/6/2018
Abstract
LIS1 mutations cause lissencephaly (LIS), a severe developmental brain malformation. Much less is known about its role in the mature nervous system. LIS1 regulates the microtubule motor cytoplasmic dynein 1 (dynein), and as LIS1 and dynein are both expressed in the adult nervous system, Lis1 could potentially regulate dynein-dependent processes such as axonal transport. We therefore knocked out Lis1 in adult mice using tamoxifen-induced, Cre-ER-mediated recombination. When an actin promoter was used to drive Cre-ER expression (Act-Cre-ER), heterozygous Lis1 knockout (KO) caused no obvious change in viability or behavior, despite evidence of widespread recombination by a Cre reporter three weeks after tamoxifen exposure. In contrast, homozygous Lis1 KO caused the rapid onset of neurological symptoms in both male and female mice. One tamoxifen-dosing regimen caused prominent recombination in the midbrain/hindbrain, PNS, and cardiac/skeletal muscle within a week; these mice developed severe symptoms in that time frame and were killed. A different tamoxifen regimen resulted in delayed recombination in midbrain/hindbrain, but not in other tissues, and also delayed the onset of symptoms. This indicates that Lis1 loss in the midbrain/hindbrain causes the severe phenotype. In support of this, brainstem regions known to house cardiorespiratory centers showed signs of axonal dysfunction in KO animals. Transport defects, neurofilament (NF) alterations, and varicosities were observed in axons in cultured DRG neurons from KO animals. Because no symptoms were observed when a cardiac specific Cre-ER promoter was used, we propose a vital role for Lis1 in autonomic neurons and implicate defective axonal transport in the KO phenotype.
Publication
Journal: Immunology
December/6/2018
Abstract
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTβ) and lymphotoxin receptor (LTβR) expression indicated that LTβR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTβR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTβR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.
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Publication
Journal: eNeuro
December/6/2018
Abstract
Neurotensin (Nts) promotes activation of dopamine (DA) neurons in the ventral tegmental area (VTA) via incompletely understood mechanisms. Nts can signal via the G protein-coupled Nts receptors 1 and 2 (NtsR1 and NtsR2), but the lack of methods to detect NtsR1- and NtsR2-expressing cells has limited mechanistic understanding of Nts action. To overcome this challenge, we generated dual recombinase mice that express FlpO-dependent Cre recombinase in NtsR1 or NtsR2 cells. This strategy permitted temporal control over recombination, such that we could identify NtsR1- or NtsR2-expressing cells and determine whether their distributions differed between the developing and adult brain. Using this system, we found that NtsR1 is transiently expressed in nearly all DA neurons and in many non-DA neurons in the VTA during development. However, NtsR1 expression is more restricted within the adult brain, where only two thirds of VTA DA neurons expressed NtsR1. By contrast, NtsR2 expression remains constant throughout lifespan, but it is predominantly expressed within glia. Anterograde tract tracing revealed that NtsR1 is expressed by mesolimbic, not mesocortical DA neurons, suggesting that VTA NtsR1 neurons may represent a functionally unique subset of VTA DA neurons. Collectively, this work reveals a cellular mechanism by which Nts can directly engage NtsR1-expressing DA neurons to modify DA signaling. Going forward, the dual recombinase strategy developed here will be useful to selectively modulate NtsR1- and NtsR2-expressing cells and to parse their contributions to Nts-mediated behaviors.
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