Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus ( Branchiostoma lanceolatum ) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis -regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Cannabigerol (CBG) is a safe non-psychotropic Cannabis-derived cannabinoid (CB), which interacts with specific targets involved in carcinogenesis. Specifically, CBG potently blocks transient receptor potential (TRP) M8 (TRPM8), activates TRPA1, TRPV1 and TRPV2 channels, blocks 5-hydroxytryptamine receptor 1A (5-HT1A) receptors and inhibits the reuptake of endocannabinoids. Here, we investigated whether CBG protects against colon tumourigenesis. Cell growth was evaluated in colorectal cancer (CRC) cells using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and 3-amino-7-dimethylamino-2-methylphenazine hydrochloride assays; apoptosis was examined by histology and by assessing caspase 3/7 activity; reactive oxygen species (ROS) production by a fluorescent probe; CB receptors, TRP and CCAAT/enhancer-binding protein homologous protein (CHOP) messenger RNA (mRNA) expression were quantified by reverse transcription-polymerase chain reaction; small hairpin RNA-vector silencing of TRPM8 was performed by electroporation. The in vivo antineoplastic effect of CBG was assessed using mouse models of colon cancer. CRC cells expressed TRPM8, CB1, CB2, 5-HT1A receptors, TRPA1, TRPV1 and TRPV2 mRNA. CBG promoted apoptosis, stimulated ROS production, upregulated CHOP mRNA and reduced cell growth in CRC cells. CBG effect on cell growth was independent from TRPA1, TRPV1 and TRPV2 channels activation, was further increased by a CB2 receptor antagonist, and mimicked by other TRPM8 channel blockers but not by a 5-HT1A antagonist. Furthermore, the effect of CBG on cell growth and on CHOP mRNA expression was reduced in TRPM8 silenced cells. In vivo, CBG inhibited the growth of xenograft tumours as well as chemically induced colon carcinogenesis. CBG hampers colon cancer progression in vivo and selectively inhibits the growth of CRC cells, an effect shared by other TRPM8 antagonists. CBG should be considered translationally in CRC prevention and cure.

In the hypothalamic arcuate nucleus (ARC), proopiomelanocortin (POMC) neurons and the POMC-derived peptide α-melanocytestimulating hormone (α-MSH) promote satiety. POMC neurons receive orexin-A (OX-A)-expressing inputs and express both OX-A receptor type 1 (OX-1R) and cannabinoid receptor type 1 (CB 1 R) on the plasma membrane. OX-A is crucial for the control of wakefulness and energy homeostasis and promotes, in OX-1R-expressing cells, the biosynthesis of the endogenous counterpart of marijuana's psychotropic and appetite-inducing component Δ 9 -tetrahydrocannabinol, i.e., the endocannabinoid 2-arachidonoylglycerol (2-AG), which acts at CB 1 R. We report that OX-A/OX-1R signaling at POMC neurons promotes 2-AG biosynthesis, hyperphagia, and weight gain by blunting α-MSH production via CB 1 R-induced and extracellularsignal-regulated kinase 1/2 activation-and STAT3 inhibitionmediated suppression of Pomc gene transcription. Because the systemic pharmacological blockade of OX-1R by SB334867 caused anorectic effects by reducing food intake and body weight, our results unravel a previously unsuspected role for OX-A in endocannabinoid-mediated promotion of appetite by combining OX-induced alertness with food seeking. Notably, increased OX-A trafficking was found in the fibers projecting to the ARC of obese mice (ob/ob and high-fat diet fed) concurrently with elevation of OX-A release in the cerebrospinal fluid and blood of mice. Furthermore, a negative correlation between OX-A and α-MSH serum levels was found in obese mice as well as in human obese subjects (body mass index > 40), in combination with elevation of alanine aminotransferase and γ-glutamyl transferase, two markers of fatty liver disease. These alterations were counteracted by antagonism of OX-1R, thus providing the basis for a therapeutic treatment of these diseases.hypocretin-1 | cannabinoid type 1 receptor | 2-arachidonoylglycerol | α-melanocyte-stimulating hormone | hypothalamus E merging anatomical, biochemical, and pharmacological evidence supports a functional interaction between endocannabinoids and orexin-A (OX-A) (also known as hypocretin-1) in the hypothalamic regulation of appetite, energy expenditure, and metabolism (1). In hypothalamic neurons, the endocannabinoid 2-arachidonylglycerol (2-AG) is under the negative control of leptin (2) and acts through the cannabinoid receptor type 1 (CB 1 R) to promote appetite by activating several intracellular pathways, including mitogen-activated protein kinases of the extracellularsignal-regulated kinase (ERK) family (3). OX-A is an orexigenic neuropeptide expressed by neurons of the lateral hypothalamus (LH), which acts through the OX-A receptor type 1 (OX-1R) (4). Activation of OX-1R by OX-A signaling has been found to affect CB 1 R function by stimulating 2-AG biosynthesis via the phospholipase C/diacylglycerol lipase α (PLC/DAGL) pathway (5) and by enhancing ERK1/2 phosphorylation and activity in cells expressing both OX-1R and CB 1 R (6, 7). Proopiomelanocortin (POMC)-containing neurons represent t...

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BACKGROUND AND PURPOSEPalmitoylethanolamide (PEA), a naturally occurring acylethanolamide chemically related to the endocannabinoid anandamide, interacts with targets that have been identified in peripheral nerves controlling gastrointestinal motility, such as cannabinoid CB1 and CB2 receptors, TRPV1 channels and PPARα. Here, we investigated the effect of PEA in a mouse model of functional accelerated transit which persists after the resolution of colonic inflammation (post-inflammatory irritable bowel syndrome). EXPERIMENTAL APPROACHIntestinal inflammation was induced by intracolonic administration of oil of mustard (OM). Mice were tested for motility and biochemical and molecular biology changes 4 weeks later. PEA, oleoylethanolamide and endocannabinoid levels were measured by liquid chromatography-mass spectrometry and receptor and enzyme mRNA expression by qRT-PCR. KEY RESULTSOM induced transient colitis and a functional post-inflammatory increase in upper gastrointestinal transit, associated with increased intestinal anandamide (but not 2-arachidonoylglycerol, PEA or oleoylethanolamide) levels and down-regulation of mRNA for TRPV1 channels. Exogenous PEA inhibited the OM-induced increase in transit and tended to increase anandamide levels. Palmitic acid had a weaker effect on transit. Inhibition of transit by PEA was blocked by rimonabant (CB1 receptor antagonist), further increased by 5′-iodoresiniferatoxin (TRPV1 antagonist) and not significantly modified by the PPARα antagonist GW6471. CONCLUSIONS AND IMPLICATIONSIntestinal endocannabinoids and TRPV1 channel were dysregulated in a functional model of accelerated transit exhibiting aspects of post-inflammatory irritable bowel syndrome. PEA counteracted the accelerated transit, the effect being mediated by CB1 receptors (possibly via increased anandamide levels) and modulated by TRPV1 channels.

While lower vertebrates contain adult stem cells (aSCs) that maintain homeostasis and drive un-exhaustive organismal growth, mammalian aSCs display mainly the homeostatic function. Here, we use lineage analysis in the medaka fish gill to address aSCs and report separate stem cell populations for homeostasis and growth. These aSCs are fate-restricted during the entire post-embryonic life and even during re-generation paradigms. We use chimeric animals to demonstrate that p53 mediates growth coordination among fate-restricted aSCs, suggesting a hierarchical organisation among lineages in composite organs like the fish gill. Homeostatic and growth aSCs are clonal but differ in their topology; modifications in tissue architecture can convert the homeostatic zone into a growth zone, indicating a leading role for the physical niche defining stem cell output. We hypothesise that physical niches are main players to restrict aSCs to a homeostatic function in animals with fixed adult size.

The current study provides strong evidence for a protective role of PEA as well as an alteration in bladder levels of PEA and of some of its targets in cyclophosphamide induced cystitis.

Yap1/Taz are well-known Hippo effectors triggering complex transcriptional programs controlling growth, survival, and cancer progression. Here we describe yap1b, a new Yap1/Taz family member with a unique transcriptional activation domain that cannot be phosphorylated by Src/Yes kinases. We show that yap1b evolved specifically in euteleosts (i.e. including medaka but not zebrafish) by duplication and adaptation of yap1. Using DamID-seq we generated maps of chromatin occupancy for Yap1, Taz (Wwtr1), and Yap1b, in gastrulating zebrafish and medaka embryos. Our comparative analyses uncover the genetic programs controlled by yap family proteins during early embryogenesis, and show largely overlapping targets for Yap1 and Yap1b. CRISPR/Cas9-induced mutation of yap1b in medaka does not result in an overt phenotype during embryogenesis or adulthood. However, yap1b mutation strongly enhances the embryonic malformations observed in yap1 mutants. Thus yap1−/−; yap1b−/− double mutants display more severe body flattening, eye misshaping, and increased apoptosis than yap1−/− single mutants; thus revealing overlapping gene functions. Our results indicate that, despite its divergent transactivation domain, Yap1b cooperates with Yap1 to regulate cell survival and tissue morphogenesis during early development.